NORMAL GENOMIC

• Coles M et al.
• Common evolutionary origin of swapped-hairpin and double-psi beta barrels.
• Structure. 2006; 14: 1489-98
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• The core of swapped-hairpin and double-psi beta barrels is formed byduplication of a conserved betaalphabeta element, suggesting a commonevolutionary origin. The path connecting the two folds is unclear as thetwo barrels are not interconvertible by a simple topological modification,such as circular permutation. We have identified a protein family whosesequence properties are intermediate to the two folds. The structure ofone of these proteins, Pyrococcus horikoshii PhS018, is also built byduplication of the conserved betaalphabeta element but shows yet a thirdtopology, which we name the RIFT barrel. This topology is widespread inthe structure database and spans three folds of the SCOP classification,including the middle domain of EF-Tu and the N domain of F1-ATPase. Wepropose that swapped-hairpin beta barrels arose from an ancestral RIFTbarrel by strand invasion and double-psi beta barrels by a strand swap. Wegroup the three barrel types into a metafold, the cradle-loop barrels.

• Gopalan G, Chopra S, Ranganathan A, Swaminathan K
• Crystal structure of uncleaved L-aspartate-alpha-decarboxylase fromMycobacterium tuberculosis.
• Proteins. 2006; 65: 796-802
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• L-aspartate-alpha-decarboxylase (ADC) is a critical regulatory enzyme inthe pantothenate biosynthetic pathway and belongs to a small class ofself-cleaving and pyruvoyl-dependent amino acid decarboxylases. Theexpression level of ADC in Mycobacterium tuberculosis (Mtb) was confirmedby cDNA analysis, immunoblotting with an anti-ADC polyclonal antibodyusing whole cell lysate and immunoelectron microscopy. The recombinant ADCproenzyme from Mycobacterium tuberculosis (MtbADC) was overexpressed in E.coli and the protein structure was determined at 2.99 A resolution. Theproteins fold into the double-psi beta-barrel structure. The subunits ofthe two tetramers (there are eight ADC molecules in the asymmetric unit)form pseudo fourfold rotational symmetry, similar to the E. coli ADCproenzyme structure. As pantothenate is synthesized in microorganisms,plants, and fungi but not in animals, structure elucidation of Mtb ADC isof substantial interest for structure-based drug development.

• van Straaten KE, Dijkstra BW, Vollmer W, Thunnissen AM
• Crystal structure of MltA from Escherichia coli reveals a unique lytictransglycosylase fold.
• J Mol Biol. 2005; 352: 1068-80
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• Lytic transglycosylases are bacterial enzymes involved in the maintenanceand growth of the bacterial cell-wall peptidoglycan. They cleave thebeta-(1,4)-glycosidic bonds in peptidoglycan forming non-reducing1,6-anhydromuropeptides. The crystal structure of the lytictransglycosylase MltA from Escherichia coli without a membrane anchor wassolved at 2.0A resolution. The enzyme has a fold completely different fromthose of the other known lytic transglycosylases. It contains two domains,the largest of which has a double-psi beta-barrel fold, similar to that ofendoglucanase V from Humicola insolens. The smaller domain also has abeta-barrel fold topology, which is weakly related to that of theRNA-binding domain of ribosomal proteins L25 and TL5. A large grooveseparates the two domains, which can accommodate a glycan strand, as shownby molecular modelling. Several conserved residues, one of which is in aposition equivalent to that of the catalytic acid of the H.insolensendoglucanase, flank this putative substrate-binding groove. Mutation ofthis residue, Asp308, abolished all activity of the enzyme, supporting thedirect participation of this residue in catalysis.

• Iyer LM, Aravind L
• The emergence of catalytic and structural diversity within the beta-clipfold.
• Proteins. 2004; 55: 977-91
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• The beta-clip fold includes a diverse group of protein domains that areunified by the presence of two characteristic waist-like constrictions,which bound a central extended region. Members of this fold includeenzymes like deoxyuridine triphosphatase and the SET methylase,carbohydrate-binding domains like the fish antifreeze proteins/Sialatesynthase C-terminal domains, and functionally enigmatic accessory subunitsof urease and molybdopterin biosynthesis protein MoeA. In this study, wereconstruct the evolutionary history of this fold using sensitive sequenceand structure comparisons methods. Using sequence profile searches, weidentified novel versions of the beta-clip fold in the bacterial flagellarchaperone FlgA and the related pilus protein CpaB, the StrU-likedehydrogenases, and the UxaA/GarD-like hexuronate dehydratases (SAFsuperfamily). We present evidence that these versions of the beta-clipdomain, like the related type III anti-freeze proteins and C-terminaldomains of sialic acid synthases, are involved in interactions withcarbohydrates. We propose that the FlgA and CpaB-like proteins mediate theassembly of bacterial flagella and Flp pili by means of their interactionswith the carbohydrate moieties of peptidoglycan. The N-terminal beta-clipdomain of the hexuronate dehydratases appears to have evolved a novelmetal-binding site, while their C-terminal domain is likely to adopt ametal-binding TIM barrel-like fold. Using structural comparisons, we showthat the beta-clip fold can be further classified into two major groups,one that includes the SAF, SET, dUTPase superfamilies, and the other thatincludes the phage lambda head decoration protein, the beta subunit ofurease and the C-terminal domain of the molybdenum cofactor biosynthesisprotein MoeA. Structural comparisons also suggest the beta-clip fold wasassembled through the duplication of a three-stranded unit. Though thethree-stranded units are likely to have had a common origin, we presentevidence that complete beta-clip domains were assembled through suchduplications, independently on multiple occasions. There is also evidencefor circular permutation of the basic three-stranded unit on differentoccasions in the evolution of the beta-clip unit. We also describe howassembly of this fold from a basic three-stranded unit has been utilizedto accommodate a variety of activities in its different versions.

• Wonderling LD, Wilkinson BJ, Bayles DO
• The htrA (degP) gene of Listeria monocytogenes 10403S is essential foroptimal growth under stress conditions.
• Appl Environ Microbiol. 2004; 70: 1935-43
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• This report describes a mutant of Listeria monocytogenes strain 10403S(serotype 1/2a) with a defective response to conditions of highosmolarity, an environment that L. monocytogenes encounters in someready-to-eat foods. A library of L. monocytogenes clones mutagenized withTn917 was generated and scored for sensitivity to 4% NaCl in order toidentify genes responsible for growth or survival in elevated-NaClenvironments. One of the L. monocytogenes Tn917 mutants, designated strainOSM1, was selected, and the gene interrupted by the transposon wassequenced. A BLAST search with the putative translated amino acid sequenceindicated that the interrupted gene product was a homolog of htrA (degP),a gene coding for a serine protease identified as a stress responseprotein in several gram-positive and gram-negative bacteria. An htrAdeletion strain, strain LDW1, was constructed, and the salt-sensitivephenotype of this strain was complemented by introduction of a plasmidcarrying the wild-type htrA gene, demonstrating that htrA is necessary foroptimal growth under conditions of osmotic stress. Additionally, strainLDW1 was tested for its response to temperature and H(2)O(2) stresses. Theresults of these growth assays indicated that strain LDW1 grew at a lowerrate than the wild-type strain at 44 degrees C but at a rate similar tothat of the wild-type strain when incubated at 4 degrees C. In addition,strain LDW1 was significantly more sensitive to a 52 degrees C heat shockthan the wild-type strain. Strain LDW1 was also defective in its responseto H(2)O(2) challenge at 37 degrees C, since 100 or 150 micro g ofH(2)O(2) was more inhibitory for the growth of strain LDW1 than for thatof the parent strain. The stress response phenotype observed for strainLDW1 is similar to that observed for other HtrA(-) organisms, whichsuggests that L. monocytogenes HtrA may play a role in degrading misfoldedproteins that accumulate under stress conditions.

• Lee BI, Suh SW
• Crystal structure of the schiff base intermediate prior to decarboxylationin the catalytic cycle of aspartate alpha-decarboxylase.
• J Mol Biol. 2004; 340: 1-7
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• l-Aspartate alpha-decarboxylase (ADC), encoded by the panD gene, catalyzesthe conversion of l-aspartate into beta-alanine. In the microorganisms,beta-alanine is required for the synthesis of pantothenate (vitamin B(5)),which is the precursor of 4'-phosphopantetheine and coenzyme A. We havedetermined the crystal structure of Helicobacter pylori ADC, a tetramericenzyme, in two forms: the apo structure at 2.0 A resolution and theisoasparagine complex structure at 1.55 A resolution. All subunits of thetetramer are self-processed at the Gly24-Ser25 linkage, producing thesmaller beta chain (residues 1-24) and the larger alpha chain (residues25-117). Each subunit contains nine beta-strands and three alpha-helices;it is folded into the double-psi beta-barrel structure. In the apostructure, the new amino terminus of the alpha chain, Ser25, is convertedinto a pyruvoyl group. In the isoasparagine complex structure, thesubstrate analog is covalently attached to the pyruvoyl group. Thisstructure represents the enzyme-substrate Schiff base intermediate thatwas proposed to form prior to the decarboxylation step in the catalyticcycle of ADC. Thus our study provides direct structural evidence for thereaction mechanism of ADC.

• Valjakka J, Rouvinen J
• Structure of 20K endoglucanase from Melanocarpus albomyces at 1.8 Aresolution.
• Acta Crystallogr D Biol Crystallogr. 2003; 59: 765-8
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• The crystal structure of the 20K endoglucanase from the thermophilicfungus Melanocarpus albomyces (Ma20k) has been determined. The structurewas refined to 1.8 A resolution using data obtained at 120 K. Ma20kbelongs to glycoside hydrolase family 45. The three-dimensional structuresof endoglucanase V (EGV) from the fungus Humicola insolens and of anendoglucanase from H. grisea var. thermoidea have previously beendetermined. The overall structure of Ma20k consists of a six-strandedbeta-barrel domain similar to that found previously in family 45endoglucanases. The flexible loop between strands V and VI, which wasdisordered in the uncomplexed structures of the Humicola endoglucanasesbut was ordered in complexed structures of EGV, is found to be wellordered in the native structure of Ma20k. The structure of Ma20k allowscomparison between thermophilic and mesophilic proteins of family 45 anddifferent principles for thermostability are discussed.

• Brunger AT, DeLaBarre B
• NSF and p97/VCP: similar at first, different at last.
• FEBS Lett. 2003; 555: 126-33
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• N-Ethylmaleimide sensitive factor (NSF) and p97/valosin-containing protein(VCP) are distantly related members of the ATPases associated with avariety of cellular activities (AAA) family of proteins. While bothproteins have been implied in cellular morphology changes involvingmembrane compartments or vesicles, more recent evidence seems to implythat NSF is primarily involved in the soluble NSF attachment receptor(SNARE)-mediated vesicle fusion by disassembling the SNARE complex whereasp97/VCP is primarily involved in the extraction of membrane proteins.These functional differences are now corroborated by major structuraldifferences based on recent crystallographic and cryo-electron microscopystudies. This review discusses these recent findings.

• Iyer LM, Koonin EV, Aravind L
• Evolutionary connection between the catalytic subunits of DNA-dependentRNA polymerases and eukaryotic RNA-dependent RNA polymerases and theorigin of RNA polymerases.
• BMC Struct Biol. 2003; 3: 1-1
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• BACKGROUND: The eukaryotic RNA-dependent RNA polymerase (RDRP) is involvedin the amplification of regulatory microRNAs during post-transcriptionalgene silencing. This enzyme is highly conserved in most eukaryotes but ismissing in archaea and bacteria. No evolutionary relationship between RDRPand other polymerases has been reported so far, hence the origin of thiseukaryote-specific polymerase remains a mystery. RESULTS: Using extensivesequence profile searches, we identified bacteriophage homologs of theeukaryotic RDRP. The comparison of the eukaryotic RDRP and their homologsfrom bacteriophages led to the delineation of the conserved portion ofthese enzymes, which is predicted to harbor the catalytic site. Further,detailed sequence comparison, aided by examination of the crystalstructure of the DNA-dependent RNA polymerase (DDRP), showed that the RDRPand the beta' subunit of DDRP (and its orthologs in archaea andeukaryotes) contain a conserved double-psi beta-barrel (DPBB) domain. ThisDPBB domain contains the signature motif DbDGD (b is a bulky residue),which is conserved in all RDRPs and DDRPs and contributes to catalysis viaa coordinated divalent cation. Apart from the DPBB domain, no similaritywas detected between RDRP and DDRP, which leaves open two scenarios forthe origin of RDRP: i) RDRP evolved at the onset of the evolution ofeukaryotes via a duplication of the DDRP beta' subunit followed bydramatic divergence that obliterated the sequence similarity outside thecore catalytic domain and ii) the primordial RDRP, which consistedprimarily of the DPBB domain, evolved from a common ancestor with the DDRPat a very early stage of evolution, during the RNA world era. The latterhypothesis implies that RDRP had been subsequently eliminated fromcellular life forms and might have been reintroduced into the eukaryoticgenomes through a bacteriophage. Sequence and structure analysis of theDDRP led to further insights into the evolution of RNA polymerases. Inaddition to the beta' subunit, beta subunit of DDRP also contains a DPBBdomain, which is, however, distorted by large inserts and does not harbora counterpart of the DbDGD motif. The DPBB domains of the two DDRPsubunits together form the catalytic cleft, with the domain from the beta'subunit supplying the metal-coordinating DbDGD motif and the one from thebeta subunit providing two lysine residues involved in catalysis. Giventhat the two DPBB domains of DDRP contribute completely different sets ofactive residues to the catalytic center, it is hypothesized that theultimate ancestor of RNA polymerases functioned as a homodimer of ageneric, RNA-binding DPBB domain. This ancestral protein probably did nothave catalytic activity and served as a cofactor for a ribozyme RNApolymerase. Subsequent evolution of DDRP and RDRP involved accretion ofdistinct sets of additional domains. In the DDRPs, these included aRNA-binding Zn-ribbon, an AT-hook-like module and a sandwich-barrel hybridmotif (SBHM) domain. Further, lineage-specific accretion of SBHM domainsand other, DDRP-specific domains is observed in bacterial DDRPs. Incontrast, the orthologs of the beta' subunit in archaea and eukaryotescontains a four-stranded alpha + beta domain that is shared with thealpha-subunit of bacterial DDRP, eukaryotic DDRP subunit RBP11,translation factor eIF1 and type II topoisomerases. The additional domainsof the RDRPs remain to be characterized. CONCLUSIONS: EukaryoticRNA-dependent RNA polymerases share the catalytic double-psi beta-barreldomain, containing a signature metal-coordinating motif, with theuniversally conserved beta' subunit of DNA-dependent RNA polymerases.Beyond this core catalytic domain, the two classes of RNA polymerases donot have common domains, suggesting early divergence from a commonancestor, with subsequent independent domain accretion. The beta-subunitof DDRP contains another, highly diverged DPBB domain. The presence of twodistinct DPBB domains in two subunits of DDRP is compatible with thehypothesis that the ith the hypothesis that the ultimate ancestor of RNApolymerases was a RNA-binding DPBB domain that had no catalytic activitybut rather functioned as a homodimeric cofactor for a ribozyme polymerase.

• Furst J, Sutton RB, Chen J, Brunger AT, Grigorieff N
• Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at11 A resolution.
• EMBO J. 2003; 22: 4365-74
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• N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family ofATPases and is involved in a number of cellular functions, includingvesicle fusion and trafficking of membrane proteins. We present thethree-dimensional structure of the hydrolysis mutant E329Q of NSFcomplexed with an ATP-ADP mixture at 11 A resolution by electroncryomicroscopy and single-particle averaging of NSF.alpha-SNAP.SNAREcomplexes. The NSF domains D1 and D2 form hexameric rings that arearranged in a double-layered barrel. Our structure is more consistent withan antiparallel orientation of the two rings rather than a parallel one.The crystal structure of the D2 domain of NSF was docked into the EMdensity map and shows good agreement, including details at the secondarystructural level. Six protrusions corresponding to the N domain of NSF(NSF-N) emerge from the sides of the D1 domain ring. The densitycorresponding to alpha-SNAP and SNAREs is located on the 6-fold axis ofthe structure, near the NSF-N domains. The density of the N domain isweak, suggesting conformational variability in this part of NSF.

• Matveeva EA, May AP, He P, Whiteheart SW
• Uncoupling the ATPase activity of the N-ethylmaleimide sensitive factor(NSF) from 20S complex disassembly.
• Biochemistry. 2002; 41: 530-6
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• The N-ethylmaleimide sensitive factor (NSF) plays a critical role inintracellular trafficking by disassembling soluble NSF attachment proteinreceptor (SNARE ) complexes. The NSF protomer consists of three domains(NSF-N, NSF-D1, and NSF-D2). Two domains (NSF-D1 and NSF-D2) contain aconserved approximately 230 amino acid cassette, which includes adistinctive motif termed the second region of homology (SRH) common to allATPases associated with various cellular activities (AAA). In hexamericNSF, several SRH residues become trans elements of the ATP binding pocket.Mutation of two conserved arginine residues in the NSF-D1 SRH (R385A andR388A) did not effect basal or soluble NSF attachment protein(SNAP)-stimulated ATPase activity; however, neither mutant underwentATP-dependent release from SNAP-SNARE complexes. A trans element of theNSF-D2 ATP binding site (K631) has been proposed to limit the ATPaseactivity of NSF-D2, but a K631D mutant retained wild-type activity. Amutation of the equivalent residue in NSF-D1 (D359K) also did not affectnucleotide hydrolysis activity but did limit NSF release from SNAP-SNAREcomplexes. These trans elements of the NSF-D1 ATP binding site (R385,R388, and D359) are not required for nucleotide hydrolysis but areimportant as nucleotide-state sensors. NSF-N mediates binding to theSNAP-SNARE complex. To identify the structural features required forbinding, three conserved residues (R67, S73, and Q76) on the surface ofNSF-N were mutated. R67E completely eliminated binding, while S73R andQ76E showed limited effect. This suggests that the surface important forSNAP binding site lies in the cleft between the NSF-N subdomains adjacentto a conserved, positively charged surface.

• Whiteheart SW, Schraw T, Matveeva EA
• N-ethylmaleimide sensitive factor (NSF) structure and function.
• Int Rev Cytol. 2001; 207: 71-112
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• Our understanding of the molecular mechanisms of membrane traffickingadvanced at a rapid rate during the 1990s. As one of the initial proteincomponents of the trafficking machinery to be identified, N-ethylmaleimidesensitive factor (NSF) has served as a reference point in many of theserecent studies. This hexameric ATPase is essential for most of themembrane-trafficking events in a cell. Initially, due to its ATPaseactivity, NSF was thought to be the motor that drove membrane fusion.Subsequent studies have shown that NSF actually plays the role of achaperone by activating SNAP receptor proteins (SNAREs) so that they canparticipate in membrane fusion. In this review we will examine the initialcharacterization of NSF, its role in membrane fusion events, and what newstructural information can tell us about NSF's mechanism of action.

• May AP, Whiteheart SW, Weis WI
• Unraveling the mechanism of the vesicle transport ATPase NSF, theN-ethylmaleimide-sensitive factor.
• J Biol Chem. 2001; 276: 21991-4

• Ofengand J et al.
• Pseudouridines and pseudouridine synthases of the ribosome.
• Cold Spring Harb Symp Quant Biol. 2001; 66: 147-59
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• psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotesall contain psi, although their number varies widely, with eukaryoteshaving the most. The small ribosomal subunit can apparently do without psiin some organisms, even though others have as many as 40 or more. Largesubunits appear to need at least one psi but can have up to 50-60. psi ismade by a set of site-specific enzymes in eubacteria, and in eukaryotes bya single enzyme complexed with auxiliary proteins andspecificity-conferring guide RNAs. The mechanism is not known in Archaea,but based on an analysis of the kinds of psi synthases found in sequencedarchaeal genomes, it is likely to involve use of guide RNAs. All psisynthases can be classified into one of four related groups, virtually allof which have a conserved aspartate residue in a conserved sequence motif.The aspartate is essential for psi formation in all twelve synthasesexamined so far. When the need for psi in E. coli was examined, the onlysynthase whose absence caused a major decrease in growth rate under normalconditions was RluD, the synthase that makes psi 1911, psi 1915, and psi1917 in the helix 69 end-loop. This growth defect was the result of amajor failure in assembly of the large ribosomal subunit. The defect couldbe prevented by supplying the rluD structural gene in trans, and also byproviding a point mutant gene that made a synthase unable to make psi.Therefore, the RluD synthase protein appears to be directly involved in50S subunit assembly, possibly as an RNA chaperone, and this activity isindependent of its ability to form psi. This result is not withoutprecedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251(E. coli numbering) in yeast mitochondria virtually blocks 50S subunitassembly and mitochondrial function (Sirum-Connolly et al. 1995), but themethylation activity of the enzyme is not required (T. Mason, pers.comm.). The absence of FtsJ, a heat shock protein that makes Um2552 in E.coli, makes the 50S subunit less stable at 1 mM Mg++ (Bugl et al. 2000)and inhibits subunit joining (Caldas et al. 2000), but, in this case, itis not yet known whether the effects are due to the lack of2'-O-methylation or to the absence of the enzyme itself. Is there any rolefor the psi residues themselves? First, as noted above, the 3 psi made byRluD which cluster in the end-loop of helix 69 are highly conserved, withone being universal (Fig. 2B). In the 70S-tRNA structure (Yusupov et al.2001), the loop of this helix containing the psi supports the anticodonarm of A-site tRNA near its juncture with the amino acid arm. The middleof helix 69 does the same thing for P-site tRNA. Unfortunately, theresolution is not yet sufficient to provide a more precise alignment ofthe psi residues with the other structural elements of the tRNA-ribosomecomplex so that one cannot yet determine what role, if any, is played bythe N-1 H that distinguishes psi from U. Second, and more generally, somepsi residues in the LSU appear to be near the site of peptide-bondformation or tRNA binding but not actually at it (Fig. 2B) (Nissen et al.2000; Yusupov et al. 2001). For example, position 2492 is commonly psi andis only six residues away from A2486, the A postulated to catalyzepeptide-bond formation. Position 2589 is psi in all the eukaryotes and isnext to 2588, which base-pairs with the C75 of A-site tRNA. Residue 2620,which interacts with the A76 of A-site-bound tRNA, is a psi or is next toa psi in eukaryotes and Archaea, and is five residues away from psi 2580in E. coli. A2637, which is between the two CCA ends of P- and A-sitetRNA, is near psi 2639, psi 2640, and psi 2641, found in a number oforganisms. Residue 2529, which contacts the backbone of A-site tRNAresidues 74-76, is near psi 2527 psi 2528 in H. marismortui. Residues2505-2507, which contact A-site tRNA residues 50-53, are near psi 2509 inhigher eukaryotes, and residues 2517-2519 in contact with A-site tRNAresidues 64-65 are within 1-3 nucleotides of psi 2520 in higher eukaryotesand psi 2514 in H. marismortui. A way to rationalize this might be toinvoke the concept suggested in the Introduction that psi acts as amolecular glue to hold loose elements in a more rigid configuration. Itmay well be that this is more important near the site of peptide-bondformation and tRNA binding, accounting for the preponderance of psi inthis vicinity. What might be the role of all the other psi in eukaryotes?One can only surmise that cells, having once acquired the ability to makepsi with guide RNAs, took advantage of the system to inexpensively placepsi wherever an undesirable loose region was found. It might be that insome of these cases, psi performs the role played by proteins in otherregions, namely that of holding the rRNA in its proper configuration.Confirmation of this hypothesis will have to await structuraldetermination of eukaryotic ribosomes.

• Brugger B et al.
• Putative fusogenic activity of NSF is restricted to a lipid mixture whosecoalescence is also triggered by other factors.
• EMBO J. 2000; 19: 1272-8
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• It has recently been reported that N-ethylmaleimide-sensitive fusionATPase (NSF) can fuse protein-free liposomes containing substantialamounts of 1,2-dioleoylphosphatidylserine (DOPS) and 1,2-dioleoyl-phosphatidyl-ethanolamine (DOPE) (Otter-Nilsson et al., 1999).The authors impart physiological significance to this observation andpropose to re-conceptualize the general role of NSF in fusion processes.We can confirm that isolated NSF can fuse liposomes of the specifiedcomposition. However, this activity of NSF is resistant to inactivation byN-ethylmaleimide and does not depend on the presence of alpha-SNAP(soluble NSF-attachment protein). Moreover, under the same conditions,either alpha-SNAP, other proteins apparently unrelated to vesiculartransport (glyceraldehyde-3-phosphate dehydrogenase or lacticdehydrogenase) or even 3 mM magnesium ions can also cause lipid mixing. Incontrast, neither NSF nor the other proteins nor magnesium had anysignificant fusogenic activity with liposomes composed of a biologicallyoccurring mixture of lipids. A straightforward explanation is that thelipid composition chosen as optimal for NSF favors non-specific fusionbecause it is physically unstable when formed into liposomes. A variety ofminor perturbations could then trigger coalescence.

• Owen DJ, Schiavo G
• A handle on NSF.
• Nat Cell Biol. 1999; 1: 1278-1278

• Kummerle R, Atta M, Scuiller J, Gaillard J, Meyer J
• Structural similarities between the N-terminal domain of Clostridiumpasteurianum hydrogenase and plant-type ferredoxins.
• Biochemistry. 1999; 38: 1938-43
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• An N-terminal domain of Clostridium pasteurianum hydrogenase I,encompassing 76 residues out of the 574 composing the full-size enzyme,had previously been overproduced in Escherichia coli and shown to form astable fold around a [2Fe-2S] cluster. This domain displays only marginalsequence similarity with [2Fe-2S] proteins of known structure, andtherefore, two-dimensional 1H NMR has been implemented to elucidatefeatures of the polypeptide fold. Despite the perturbing presence of theparamagnetic [2Fe-2S] cluster, 57 spin systems were detected in the TOCSYspectra, 52 of which were sequentially assigned through NOEconnectivities. Several secondary structure elements were identified. TheN terminus of the protein consists of two antiparallel beta strandsfollowed by an alpha helix contacting both strands. Two additionalantiparallel beta strands, one of them at the C terminus of the sequence,form a four-stranded beta sheet together with the two N-terminal strands.The proton resonances that can be attributed to this beta2alphabeta2structural motif undergo no paramagnetic perturbations, suggesting that itis distant from the [2Fe-2S] cluster. In plant- and mammalian-typeferredoxins, a very similar structural pattern is found in the part of theprotein farthest from the [2Fe-2S] cluster. This indicates that theN-terminal domain of C. pasteurianum hydrogenase folds in a manner verysimilar to those of plant- and mammalian-type ferredoxins over asignificant part (ca. 50%) of its structure. Even in the vicinity of themetal site, where 1H NMR data are blurred by paramagnetic interactions,the N-terminal domains of hydrogenase and mammalian- and plant-typeferredoxins most likely display significant structural similarity, asinferred from local sequence alignments and from previously reportedcircular dichroism and resonance Raman spectra. These data affordstructural information on a kind of [2Fe-2S] cluster-containing domainthat occurs in a number of redox enzymes and complexes. In addition,together with previously published sequence alignments, they highlight thewidespread distribution of the plant-type ferredoxin fold in bioenergeticsystems encompassing anaerobic metabolism, photosynthesis, and aerobicrespiratory chains.

• Neuwald AF
• The hexamerization domain of N-ethylmaleimide-sensitive factor: structuralclues to chaperone function.
• Structure. 1999; 7: 1923-1923
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• The hexameric structure of the D2 ATP-binding module ofN-ethylmaleimide-sensitive factor (NSF), a chaperone involved in SNAREcomplex disassembly, was recently determined. This structure and thepreviously determined structure of the DNA polymerase III delta' subunithave far-reaching biological significance because these modules arerelated to diverse ATPases that promote the assembly, disassembly andoperation of various protein complexes.

• Fleming KG et al.
• A revised model for the oligomeric state of the N-ethylmaleimide-sensitivefusion protein, NSF.
• J Biol Chem. 1998; 273: 15675-81
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• The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase thatplays an essential role in intracellular membrane trafficking. Previousreports have concluded that NSF forms either a tetramer or a trimer insolution, and that assembly of the oligomer is essential for efficientactivity in membrane transport reactions. However, in recent electronmicroscopic analyses NSF appears as a hexagonal cylinder similar in sizeto related ATPases known to be hexamers. We have therefore reevaluatedNSF's oligomeric state using a variety of quantitative biophysicaltechniques. Sedimentation equilibrium and sedimentation velocityanalytical ultracentrifugation, transmission electron microscopy withrotational image analysis, scanning transmission electron microscopy, andmultiangle light scattering all demonstrate that, in the presence ofnucleotide, NSF is predominantly a hexamer. Sedimentation equilibriumresults further suggest that the NSF hexamer is held together byoligomerization of its D2 domains. The sedimentation coefficient, s20,w0,of 13.4 (+/-0. 1) S indicates that NSF has unusual hydrodynamiccharacteristics that cannot be solely explained by its shape. Thedemonstration that NSF is a hexameric oligomer highlights structuralsimilarities between it and several related ATPases which act by switchingthe conformational states of their protein substrates in order to activatethem for subsequent reactions.

• Sommer P, Bormann C, Gotz F
• Genetic and biochemical characterization of a new extracellular lipasefrom Streptomyces cinnamomeus.
• Appl Environ Microbiol. 1997; 63: 3553-60
• Display abstract

• Streptomyces cinnamomeus Tu89 secretes a 30-kDa esterase and a 50-kDalipase. The lipase-encoding gene, lipA, was cloned from genomic DNA intoStreptomyces lividans TK23 with plasmid vector pIJ702. Two lipase-positiveclones were identified; each recombinant plasmid had a 5.2-kb MboI insertthat contained the complete lipA gene. The two plasmids differed in theorientation of the insert and the degree of lipolytic activity produced.The lipA gene was sequenced; lipA encodes a proprotein of 275 amino acids(29,213 Da) with a pI of 5.35. The LipA signal peptide is 30 amino acidslong, and the mature lipase sequence is 245 amino acids long (26.2 kDa)and contains six cysteine residues. The conserved catalytic serine residueof LipA is in position 125. Sequence similarity of the mature lipases (29%identity, 60% similarity) was observed mainly in the N-terminal 104 aminoacids with the group II Pseudomonas lipases; no similarity to the twoStreptomyces lipase sequences was found. lipA was also expressed inEscherichia coli under the control of lacZ promoter. In the presence ofthe inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), growth of theE. coli clone was severely affected, and the cells lysed in liquid medium.Lipase activity in the E. coli clone was found mainly in the pelletfraction. In sodium dodecyl sulfate-polyacrylamide gel electrophoresisanalysis, three additional protein bands of 50, 29, and 27 kDa werevisible. The 27-kDa protein showed lipolytic activity and represents themature lipase; the 29- and 50-kDa forms showed no activity and veryprobably represent the unprocessed form and a dimeric misfolded form,respectively. For higher expression of lipA in S. lividans, the gene wascloned next to the strong aphII promoter. In contrast to thelipA-expressing E. coli clone, S. cinnamomeus and the corresponding S.lividans clone secreted only an active protein of 50 kDa. The lipaseshowed highest activity with C6 and C18 triglycerides; no activity wasobserved with phospholipids, Tween 20, or p-nitrophenylesters. Upstream oflipA and in the same orientation, an open reading frame, orfA, is foundwhose deduced protein sequence (519 amino acids) shows similarity tovarious membrane-localized transporters. Downstream of lipA and in theopposite orientation, an open reading frame, orfB (encoding a199-amino-acid protein) is found, which shows no conspicuous sequencesimilarity to known proteins, other than an NAD and flavin adeninedinucleotide binding-site sequence.

• Laskowska E, Kuczynska-Wisnik D, Skorko-Glonek J, Taylor A
• Degradation by proteases Lon, Clp and HtrA, of Escherichia coli proteinsaggregated in vivo by heat shock; HtrA protease action in vivo and invitro.
• Mol Microbiol. 1996; 22: 555-71
• Display abstract

• Thermally aggregated, endogenous proteins of Escherichia coli form adistinct fraction, denoted S, which is separable bysucrose-density-gradient centrifugation. It was shown earlier that DnaK,DnaJ, IbpA and IbpB heat-shock proteins are associated with the Sfraction. Comparison of the rise and decay of the S fraction in mutantsdefective for heat-shock proteases Lon (La), Clp, HtrA (DegP, Do) and inwild-type strains made studies of proteolysis and the function of theheat-shock response possible in vivo. Different timing and the extent ofaction of particular proteases was revealed by the initial size and decaykinetics of the S fraction. The proteases Lon, Clp, and HtrA allparticipated in removal of the aggregated proteins. Mutation in the geneencoding ClpB caused the most prominent effect (47% stabilization of the Sfraction). The correlation between the disappearance of the S fraction andproteolytic activity was supported by the result of the in vitro reaction.Approximately one third of the isolated S fraction was converted totrichloroacetic acid-soluble products by the purified HtrA protease. Mg2+ions stimulated the reaction, in contrast to the reaction of the HtrAprotease with casein. The digestion of the aggregated proteins, unlike thedigestion of casein, by HtrA protease in vitro was inhibited by addedDnaJ, which might reflect protection of the aggregated proteins in vivo byDnaJ from excessive degradation. One might expect that such an activity ofDnaJ would promote denatured protein renaturation versus proteolysis.Moreover, among the aggregated proteins that are discernible byelectrophoresis, none could be identified as being more susceptible thanany other to HtrA degradation. The separation pattern of these proteinsbefore and after the in vitro digestion did not show a differencecorresponding to the loss of about 30% of constituting proteins. This wasinterpreted as recognition by the HtrA protease of a state of proteindenaturation rather than specific amino acid sequences in particularproteins. We conclude that the fraction consisting of proteinsheat-aggregated in vivo (i.e. the S fraction) contains endogenoussubstrates for the heat-shock proteases tested. Their use for in vitroreaction reveals information that is in some respects different from thatobtained with exogenous substrates such as casein.

• Shang ES, Summers TA, Haake DA
• Molecular cloning and sequence analysis of the gene encoding LipL41, asurface-exposed lipoprotein of pathogenic Leptospira species.
• Infect Immun. 1996; 64: 2322-30
• Display abstract

• We report the cloning of the gene encoding a surface-exposed leptospirallipoprotein, designated LipL41. In a previous study, a 41-kDa proteinantigen was identified on the surface of Leptospira kirschneri (D. A.Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A.Lovett, Infect. Immun. 59:1131-1140, 1991). We obtained the N-terminalamino acid sequence of a staphylococcal V8 proteolytic-digest fragment inorder to design an oligonucleotide probe.A Lambda ZAP II librarycontaining EcoRI fragments of L. kirschneri DNA was screened, and a 2.3-kbDNA fragment which contained the entire structural lipL41 gene wasidentified. The deduced amino acid sequence of LipL41 would encode a355-amino-acid polypeptide with a 19-amino-acid signal peptide, followedby an L-X-Y-C lipoprotein signal peptidase cleavage site. A recombinantHis6-LipL41 fusion protein was expressed in Escherichia coli in order togenerate specific rabbit antiserum. LipL41 is solubilized by Triton X-114extraction of L. kirschneri; phase separation results in partitioning ofLipL41 exclusively into the detergent phase. At least eight proteins,including LipL41 and the other major Triton X-114 detergent phaseproteins, are intrinsically labeled during incubation of L. kirschneri inmedia containing [3H] palmitate. Processing of LipL41 is inhibited byglobomycin, a selective inhibitor of lipoprotein signal peptidase. TritonX-100 extracts of L. kirschneri contain immunoprecipitable OmpL1 (porin),LipL41, and another lipoprotein, LipL36. However, in contrast to LipL36,only LipL41 and OmpL1 were exposed on the surface of intact organisms.Immunoblot analysis of a panel of Leptospira species reveals that LipL41expression is highly conserved among leptospiral pathogens.

• Waller PR, Sauer RT
• Characterization of degQ and degS, Escherichia coli genes encodinghomologs of the DegP protease.
• J Bacteriol. 1996; 178: 1146-53
• Display abstract

• The degQ and degS genes of Escherichia coli encode proteins of 455 and 355residues, respectively, which are homologs of the DegP protease. Thepurified DegQ protein has the properties of a serine endoprotease and isprocessed by the removal of a 27-residue amino-terminal signal sequence. Aplasmid expressing degQ rescues the temperature-sensitive phenotype of astrain bearing the degP41 deletion, implying that DegQ, like DegP,functions as a periplasmic protease in vivo. Deletions in the degQ genecause no obvious growth defect, while those in the degS gene result in asmall-colony phenotype. The latter phenotype is rescued by a plasmidexpressing the degS gene but not by plasmids expressing the degQ or degPgenes. This result and the inability of a plasmid expressing degS torescue the temperature-sensitive degP41 phenotype indicate that the DegSprotein is functionally different from the DegQ and DegP proteins.

• Boucher JC, Martinez-Salazar J, Schurr MJ, Mudd MH, Yu H, Deretic V
• Two distinct loci affecting conversion to mucoidy in Pseudomonasaeruginosa in cystic fibrosis encode homologs of the serine protease HtrA.
• J Bacteriol. 1996; 178: 511-23
• Display abstract

• Conversion to a mucoid, exopolysaccharide alginate-overproducing phenotypein Pseudomonas aeruginosa is associated with chronic respiratoryinfections in cystic fibrosis. Mucoidy is caused by muc mutations thatderepress the alternative sigma factor AlgU, which in turn activatesalginate biosynthetic and ancillary regulatory genes. Here we report themolecular characterization of two newly identified genes, algW and mucD,that affect expression of mucoidy. The algW gene, mapping at 69 min, wasisolated on the basis of its ability to suppress mucoidy and reducetranscription of the alginate biosynthetic gene algD. The predictedprimary structure of AlgW displayed similarity to HtrA (DegP), a serineprotease involved in proteolysis of abnormal proteins and required forresistance to oxidative and heat stress in enteric bacteria. Inactivationof algW on the chromosome of the wild-type nonmucoid strain PAO1 causedincreased sensitivity to heat, H2O2, and paraquat, a redox cyclingcompound inducing intracellular levels of superoxide. This mutation alsopermitted significant induction of alginate production in the presence ofsubinhibitory concentrations of paraquat. Two new genes, mucC and mucD,were identified immediately downstream of the previously characterizedportion (algU mucA mucB) of the gene cluster at 67.5 min encoding thealternative sigma factor AlgU and its regulators. Interestingly, thepredicted gene product of mucD also showed similarities to HtrA.Inactivation of mucD on the PAO1 chromosome resulted in conversion to themucoid phenotype. The mutation in mucD also caused increased sensitivityto H2O2 and heat killing. However, in contrast to algW mutants, noincrease in susceptibility to paraquat was observed in mucD mutants. Thesefindings indicate that algW and mucD play partially overlapping butdistinct roles in P. aeruginosa resistance to reactive oxygenintermediates and heat. In addition, since mutations in mucD and algWcause conversion to mucoidy or lower the threshold for its induction byreactive oxygen intermediates, these factors may repress alginatesynthesis either directly by acting on AlgU or its regulators orindirectly by removing physiological signals that may activate this stressresponse system.

• Witke C, Gotz F
• Cloning and nucleotide sequence of the signal peptidase II (lsp)-gene fromStaphylococcus carnosus.
• FEMS Microbiol Lett. 1995; 126: 233-9
• Display abstract

• Staphylococcus carnosus TM300 is able to synthesize at least sevenlipoproteins with molecular masses between 15 and 45 kDa; the proteins arelocated in the membrane fraction. It can be concluded that this strainalso posesses the enzymes involved in lipoprotein modification andprolipoprotein signal peptidase (signal peptidase II) processing. The geneencoding the prolipoprotein signal peptidase, lsp, from Staphylococcuscarnosus TM300 was cloned in Escherichia coli and sequenced. The deducedamino acid sequence of the Lsp showed amino acid similarities with theLsp's of S. aureus, Enterobacter aerogenes, E. coli, and Pseudomonasfluorescens. The hydropathy profile reveals four hydrophobic segmentswhich are homologous to the putative transmembrane regions of the E. colisignal peptidase II. E. coli strains carrying lsp of S. carnosus exhibitedan increased globomycin resistance.

• Snyder WB, Davis LJ, Danese PN, Cosma CL, Silhavy TJ
• Overproduction of NlpE, a new outer membrane lipoprotein, suppresses thetoxicity of periplasmic LacZ by activation of the Cpx signal transductionpathway.
• J Bacteriol. 1995; 177: 4216-23
• Display abstract

• The LamB-LacZ-PhoA tripartite fusion protein is secreted to the periplasm,where it exerts a toxicity of unknown origin during high-level synthesisin the presence of the inducer maltose, a phenotype referred to as maltosesensitivity. We selected multicopy suppressors of this toxicity that allowgrowth of the tripartite fusion strains in the presence of maltose.Mapping and subclone analysis of the suppressor locus identified apreviously uncharacterized chromosomal region at 4.7 min that isresponsible for suppression. DNA sequence analysis revealed a new genewith the potential to code for a protein of 236 amino acids with apredicted molecular mass of 25,829 Da. The gene product contains anamino-terminal signal sequence to direct the protein for secretion and aconsensus lipoprotein modification sequence. As predicted from thesequence, the suppressor protein is labeled with [3H]palmitate and islocalized to the outer membrane. Accordingly, the gene has been named nlpE(for new lipoprotein E). Increased expression of NlpE suppresses themaltose sensitivity of tripartite fusion strains and also theextracytoplasmic toxicities conferred by a mutant outer membrane protein,LamBA23D. Suppression occurs by activation of the Cpx two-component signaltransduction pathway. This pathway controls the expression of theperiplasmic protease DegP and other factors that can combat certain typesof extracytoplasmic stress.

• Bishop RE, Penfold SS, Frost LS, Holtje JV, Weiner JH
• Stationary phase expression of a novel Escherichia coli outer membranelipoprotein and its relationship with mammalian apolipoprotein D.Implications for the origin of lipocalins.
• J Biol Chem. 1995; 270: 23097-103
• Display abstract

• We report a novel outer membrane lipoprotein of Escherichia coli. DNAsequencing between ampC and sugE at the 94.5 min region of the E. colichromosome revealed an open reading frame specifying 177 amino acidresidues. Primer extension analysis demonstrated that the promoter isactivated at the transition between exponential and stationary growthphases under control of the rpoS sigma factor gene, and this was confirmedin vivo by monitoring expression of beta-galactosidase activity from alacZ translational fusion. The amino acid sequence exhibited 31% identitywith human apolipoprotein D (apoD), which is a component of plasma highdensity lipoprotein and belongs to the eukaryotic family of lipocalins.The bacterial lipocalin (Blc) contained a short deletion of 7 amino acidresidues corresponding to a hydrophobic surface loop that is thought tofacilitate the physical interaction between apoD and high densitylipoprotein. However, Blc exhibited a typical prokaryotic lipoproteinsignal peptide at its amino terminus. Overexpression, membranefractionation, and metabolic labeling with [3H]palmitate demonstrated thatBlc is indeed a globomycin-sensitive outer membrane lipoprotein. Blcrepresents the first bacterial member of the family of lipocalins and mayserve a starvation response function in E. coli.

• Zuber M, Hoover TA, Court DL
• Analysis of a Coxiella burnetti gene product that activates capsulesynthesis in Escherichia coli: requirement for the heat shock chaperoneDnaK and the two-component regulator RcsC.
• J Bacteriol. 1995; 177: 4238-44
• Display abstract

• A 1.2-kb EcoRI genomic DNA fragment of Coxiella burnetti, when cloned ontoa multicopy plasmid, was found to induce capsule synthesis (mucoidy) inEscherichia coli. Nucleotide sequence analysis revealed the presence of anopen reading frame that could encode a protein of 270 amino acids.Insertion of a tet cassette into a unique NruI restriction site resultedin the loss of induction of mucoidy. Because of its ability to inducemucoidy, we designated this gene mucZ. Computer search for homologies tomucZ revealed 42% identity to an open reading frame located at 1 min ofthe E. coli chromosome. Interestingly, the C-terminal amino acid residuesof MucZ share significant homology with the J domain of the DnaJ proteinand its homologs, suggesting potential interactions between MucZ andcomponents of the DnaK-chaperone machinery. Results presented in thispaper suggest that E. coli requires DnaK-chaperone machinery forLon-RcsA-mediated induction of capsule synthesis, as noticed first by S.Gottesman (personal communication). The induction caused by MucZ isindependent of Lon-RcsA and is mediated through the two-componentregulators RcsC and RcsB. DnaK and GrpE but not DnaJ are also required forthe RcsB-mediated MucZ induction, and we propose that MucZ is a DnaJ-likechaperone protein that might be required for the formation of an activeRcsA-RcsB complex and for the RcsC-dependent phosphorylation of RcsB.Discussions are presented that suggest three different roles foralternative forms of the DnaK-chaperone machinery in capsule production.

• Ichikawa JK, Li C, Fu J, Clarke S
• A gene at 59 minutes on the Escherichia coli chromosome encodes alipoprotein with unusual amino acid repeat sequences.
• J Bacteriol. 1994; 176: 1630-8
• Display abstract

• We report a 1.432-kb DNA sequence at 59 min on the Escherichia colichromosome that connects the published sequences of the pcm gene for theisoaspartyl protein methyltransferase and that of the katF or rpoS(katF/rpoS) gene for a sigma factor involved in stationary-phase geneexpression. Analysis of the DNA sequence reveals an open reading framepotentially encoding a polypeptide of 379 amino acids. The polypeptidesequence includes a consensus bacterial lipidation sequence present atresidues 23 to 26 (Leu-Ala-Gly-Cys), four octapeptide proline- andglutamine-rich repeats of consensus sequence QQPQIQPV, and fourheptapeptide threonine- and serine-rich repeats of consensus sequencePTA(S,T)TTE. The deduced amino acid sequence, especially in the C-terminalregion, is similar to that of the Haemophilus somnus LppB lipoproteinouter membrane antigen (40% overall sequence identity; 77% identity inlast 95 residues). The LppB lipoprotein binds Congo red dye and has beenproposed to be a virulence determinant in H. somnus. Utilizing a plasmidconstruct with the E. coli gene under the control of a phage T7 promoter,we demonstrate the lipidation of this gene product by the incorporation of[3H]palmitic acid into a 42-kDa polypeptide. We also show that treatmentof E. coli cells with globomycin, an inhibitor of the lipoprotein signalpeptidase, results in the accumulation of a 46-kDa precursor. We thusdesignate the protein NlpD (new lipoprotein D). E. coli cellsoverexpressing NlpD bind Congo red dye, suggesting a common function withthe H. somnus LppB protein. Disruption of the chromosomal E. coli nlpDgene by insertional mutagenesis results in decreased stationary-phasesurvival after 7 days.

• Yamanaka K, Mitani T, Ogura T, Niki H, Hiraga S
• Cloning, sequencing, and characterization of multicopy suppressors of amukB mutation in Escherichia coli.
• Mol Microbiol. 1994; 13: 301-12
• Display abstract

• The mukB gene codes for a 177 kDa protein, which might be a candidate fora force-generating enzyme in chromosome positioning in Escherichia coli.The mukB106 mutant produces normal-sized, anucleate cells and shows atemperature-sensitive colony formation. To identify proteins interactingwith the MukB protein, we isolated three multicopy suppressors (msmA,msmB, and msmC) to the temperature-sensitive colony formation of themukB106 mutation. The msmA gene, which could not suppress the productionof anucleate cells, was found to be identical to the dksA gene. The msmBand msmC genes suppressed the production of anucleate cells as well as thetemperature-sensitive colony formation. However, none of them couldsuppress both phenotypes in a mukB null mutation. DNA sequencing revealedthat the msmB gene was identical to the cspC gene and that the msmC genehad not been described before. A homology search revealed that the aminoacid sequences of both MsmB and MsmC possessed high similarity to proteinscontaining the cold-shock domain, such as CspA of E. coli and the Y-boxbinding proteins of eukaryotes; this suggests that MsmB and MsmC might beDNA-binding proteins that recognize the CCAAT sequence. Hence, the msmBand msmC genes were renamed cspC and cspE, respectively. Possiblemechanisms for suppression of the mukB106 mutation are discussed.

• Seiffer D, Klein JR, Plapp R
• EnvC, a new lipoprotein of the cytoplasmic membrane of Escherichia coli.
• FEMS Microbiol Lett. 1993; 107: 175-8
• Display abstract

• A gene product with an apparent molecular mass of approximately 39,000 Dacan be identified in the cytoplasmic membrane of Escherichia coli uponexpression of cloned envC. In this communication we report that theproduct was labelled with [3H]glycerol and [3H]palmitic acid, and aprecursor molecule of increased molecular mass was accumulated when cellswere treated with globomycin, a specific inhibitor for the prolipoproteinsignal peptidase. The same precursor molecule was encoded by an envCmutant gene, in which the cysteine residue in a pentapeptide sequence,Leu-Ile-Ala-Gly-Cys24 within the amino terminal region of EnvC, wasreplaced by tryptophane (Trp24). This protein was not labelled with[3H]glycerol. The results demonstrate that the envC gene productrepresents a new lipoprotein of the cytoplasmic membrane of E. coli.

• Hazes B, Hol WG
• Comparison of the hemocyanin beta-barrel with other Greek keybeta-barrels: possible importance of the "beta-zipper" in proteinstructure and folding.
• Proteins. 1992; 12: 278-98
• Display abstract

• The Greek key beta-barrel topology is a folding motif observed in manyproteins of widespread evolutionary origin. The arthropodan hemocyaninsalso have such a Greek key beta-barrel, which forms the core of the thirddomain of this protein. The hemocyanin beta-barrel was found to bestructurally very similar to the beta-barrels of the immunoglobulindomains, Cu,Zn-superoxide dismutase and the chromophore carrying antitumorproteins. The structural similarity within this group of protein familiesis not accompanied by an evolutionary or functional relationship. It istherefore possible to study structure-sequence relations without bias fromnonstructural constraints. The present study reports a conserved patternof features in these Greek key beta-barrels that is strongly suggestive ofa folding nucleation site. This proposed nucleation site, which we call a"beta-zipper," shows a pattern of well-conserved, large hydrophobicresidues on two sequential beta-strands joined by a short loop. Eachbeta-zipper strand is near the center of one of the beta-sheets, so thatthe two strands face each other from opposite sides of the barrel andinteract through their hydrophobic side chains, rather than forming ahydrogen-bonded beta-hairpin. Other protein families with Greek keybeta-barrels that do not as strongly resemble the immunoglobulinfold--such as the azurins, plastocyanins, crystallins, andprealbumins--also contain the beta-zipper pattern, which might thereforebe a universal feature of Greek key beta-barrel proteins.

• Baird L, Lipinska B, Raina S, Georgopoulos C
• Identification of the Escherichia coli sohB gene, a multicopy suppressorof the HtrA (DegP) null phenotype.
• J Bacteriol. 1991; 173: 5763-70
• Display abstract

• We cloned and sequenced the sohB gene of Escherichia coli. Thetemperature-sensitive phenotype of bacteria that carry a Tn10 insertion inthe htrA (degP) gene is relieved when the sohB gene is present in the cellon a multicopy plasmid (30 to 50 copies per cell). The htrA gene encodes aperiplasmic protease required for bacterial viability only at hightemperature, i.e., above 39 degrees C. The sohB gene maps to 28 min on theE. coli chromosome, precisely between the topA and btuR genes. The geneencodes a 39,000-Mr precursor protein which is processed to a 37,000-Mrmature form. Sequencing of a DNA fragment containing the gene revealed anopen reading frame which could encode a protein of Mr 39,474 with apredicted signal sequence cleavage site between amino acids 22 and 23.Cleavage at this site would reduce the size of the processed protein to37,474 Mr. The predicted protein encoded by the open reading frame hashomology with the inner membrane enzyme protease IV of E. coli, whichdigests cleaved signal peptides. Therefore, it is possible that the sohBgene encodes a previously undiscovered periplasmic protease in E. colithat, when overexpressed, can partially compensate for the missing HtrAprotein function.

• Bouvier J, Pugsley AP, Stragier P
• A gene for a new lipoprotein in the dapA-purC interval of the Escherichiacoli chromosome.
• J Bacteriol. 1991; 173: 5523-31
• Display abstract

• Cloning and sequence analysis of the region located downstream of the dapAgene of Escherichia coli has revealed the presence of an open readingframe that is cotranscribed with dapA. This gene codes for a344-amino-acid polypeptide with a potential signal sequence characteristicof a lipoprotein. When this gene, called nlpB, is expressed from amulticopy plasmid in bacteria grown in the presence of [3H]palmitate, alabeled 37-kDa protein is produced. A slightly larger precursor moleculeis detected when minicells expressing nlpB are treated with globomycin, aspecific inhibitor of lipoprotein signal peptidase. Therefore, the nlpBgene encodes a new lipoprotein, designated NlpB. This lipoprotein isdetected in outer membrane vesicles prepared from osmotically lysedspheroplasts and appears to be nonessential, since a strain in which thenlpB gene is disrupted by insertion of a chloramphenicol resistance geneis still able to grow and shows no discernible NlpB phenotype. Theputative transcription termination signals of the dapA-nlpB operon overlapthe promoter of the adjacent purC gene.

• Lipinska B, Zylicz M, Georgopoulos C
• The HtrA (DegP) protein, essential for Escherichia coli survival at hightemperatures, is an endopeptidase.
• J Bacteriol. 1990; 172: 1791-7
• Display abstract

• As a preliminary step in the understanding of the function of theEscherichia coli HtrA (DegP) protein, which is indispensable for bacterialsurvival only at elevated temperatures, the protein was purified andpartially characterized. The HtrA protein was purified from cells carryingthe htrA gene cloned into a multicopy plasmid, resulting in itsoverproduction. The sequence of the 13 N-terminal amino acids of thepurified HtrA protein was determined and was identical to the onepredicted for the mature HtrA protein by the DNA sequence of the clonedgene. Moreover, the N-terminal sequence showed that the 48-kilodalton HtrAprotein is derived by cleavage of the first 26 amino acids of the pre-HtrAprecursor polypeptide and that the point of cleavage follows a typicaltarget sequence recognized by the leader peptidase enzyme. The HtrAprotein was shown to be a specific endopeptidase which was inhibited bydiisopropylfluorophosphate, suggesting that HtrA is a serine protease.

• Kang PJ, Craig EA
• Identification and characterization of a new Escherichia coli gene that isa dosage-dependent suppressor of a dnaK deletion mutation.
• J Bacteriol. 1990; 172: 2055-64
• Display abstract

• We report the isolation and characterization of a previously unidentifiedEscherichia coli gene that suppresses the temperature-sensitive growth andfilamentation of a dnaK deletion mutant strain. Introduction of amulticopy plasmid carrying this wild-type gene into a dnaK deletion mutantstrain rescued the temperature-sensitive growth of the dnaK deletionmutant strain at 40.5 degrees C and the filamentation, fully at 37 degreesC and partially at 40.5 degrees C. However, the inability of dnaK mutantcells to support bacteriophage lambda growth was not suppressed. This genewas also able to suppress the temperature-sensitive growth of a grpE280mutant strain at 41 degrees C. Filamentation of the grpE280 mutant strainwas suppressed at 37 degrees C but not at 41 degrees C. The dnaKsuppressor gene, designated dksA, maps near the mrcB gene (3.7 min on theE. coli chromosome). DNA sequence analysis and in vivo experiments showedthat dksA encodes a 17,500-Mr polypeptide. Gene disruption experimentsindicated that dksA is not an essential gene.

• Doi M et al.
• Determinations of the DNA sequence of the mreB gene and of the geneproducts of the mre region that function in formation of the rod shape ofEscherichia coli cells.
• J Bacteriol. 1988; 170: 4619-24
• Display abstract

• The 6.5-kilobase mre region at 71 min in the Escherichia coli chromosomemap, where genes involved in formation of a rod-shaped cell form a genecluster, was analyzed by in vivo protein synthesis in a maxicell systemand by base sequencing of DNA. An open reading frame that may code for aprotein with an Mr of about 37,000 on sodium dodecylsulfate-polyacrylamide gels was found and was correlated with the mreBgene. N-terminal amino acid sequencing of the hybrid mreB-lacZ proteinconfirmed the production by mreB of a protein of 347 amino acid residueswith a molecular weight of 36,958. The amino acid sequence of this proteindeduced from the DNA sequence showed close similarity with that of aprotein of the ftsA gene which is involved in cell division of E. coli.Three other contiguous genes that formed three proteins with Mrs of about40,000, 22,000, and 51,000, respectively, were detected downstream of themreB gene by in vivo protein synthesis. The mreB protein and some of thesethree proteins may function together in determination of cell shape.

• Chen R, Henning U
• Nucleotide sequence of the gene for the peptidoglycan-associatedlipoprotein of Escherichia coli K12.
• Eur J Biochem. 1987; 163: 73-7
• Display abstract

• During attempts to clone the gene for the Escherichia coli outer membraneprotein III another gene was recovered. Its nucleotide sequence wasdetermined and the deduced amino acid sequence showed that the gene doesnot encode protein III. It codes for a 173-residue polypeptide; 21NH2-terminal residues are typical for a signal peptide. The sequencearound the putative site (Ala-Cys) for removing this peptide,Ala-Ile-Ala-Ala-Cys-Ser-Ser-Asn, is highly homologous to that of the majorcell envelope lipoprotein (Braun lipoprotein) surrounding its processingsite; it is also homologous to the consensus pentapeptideLeu-Leu-Ala-Gly-Cys present in other lipoproteins of gram-negativebacteria. It could be shown that the gene expresses a lipoprotein with allthe properties, including the amino acid composition, of thepeptidoglycan-associated lipoprotein (PAL) [Mizuno, T. (1979) J. Biochem.(Tokyo) 86, 991-1000]. Therefore, the cloned gene is the pal gene. Theprotein does not contain hydrophobic regions which would serve as amembrane anchor. Tandemly repeated amino acid sequences exist at and nearthe NH2-terminus of the mature protein which are homologous to suchrepeats in the Braun lipoprotein, suggesting a common origin of this partof the two proteins. Attempts to place a transposon into the pal gene wereunsuccessful. Hence the complete absence of the protein may be lethal andits function remains unknown.

• Yu F, Inouye S, Inouye M
• Lipoprotein-28, a cytoplasmic membrane lipoprotein from Escherichia coli.Cloning, DNA sequence, and expression of its gene.
• J Biol Chem. 1986; 261: 2284-8
• Display abstract

• Escherichia coli contains several lipoproteins in addition to the majorouter membrane lipoprotein (Ichihara, S., Hussain, M., and Mizushima, S.(1981) J. Biol. Chem. 256, 3125-3129). We cloned the gene for one of thesenew lipoproteins by using a synthetic 15-mer oligonucleotide probeidentical to the DNA sequence at the signal peptide cleavage site of themajor lipoprotein. The DNA sequence of the cloned gene revealed an openreading frame encoding a 272-amino acid protein with a signal peptide of23 amino acid residues. The amino acid sequence of the putative cleavagesite region of the signal peptide, -Leu-Leu-Ala-Gly-Cys-, is identical tothat of the major lipoprotein. When the cloned gene was expressed in E.coli, a gene product with an apparent molecular weight of approximately29,000 was identified which agrees well with the calculated molecularweight (27,800). The product was labeled with [3H]glycerol, and aprecursor molecule of increased molecular weight was accumulated whencells were treated with globomycin, a specific inhibitor forprolipoprotein signal peptidase. We thus designed the gene product aslipoprotein-28. Unlike the major lipoprotein, lipoprotein-28 was found tobe localized in the cytoplasmic membrane. A possible orientation oflipoprotein-28 in the E. coli envelope is discussed.