NORMAL GENOMIC

• Dedieu L, Serveau-Avesque C, Canaan S
• Identification of residues involved in substrate specificity and cytotoxicity of two closely related cutinases from Mycobacterium tuberculosis.
• PLoS One. 2013; 8: 66913-66913
• Display abstract

• The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.

• Perumalla SR, Sun CC
• Confused HCl: hydrogen chloride or hydrochloric acid?
• Chemistry. 2012; 18: 6462-4

• Dolot R, Ozga M, Krakowiak A, Nawrot B
• High-resolution X-ray structure of the rabbit histidine triad nucleotide-binding protein 1 (rHINT1)-adenosine complex at 1.10 A resolution.
• Acta Crystallogr D Biol Crystallogr. 2011; 67: 601-7
• Display abstract

• Histidine triad nucleotide-binding protein 1 (HINT1) represents the most ancient and widespread branch in the histidine-triad protein superfamily. HINT1 plays an important role in various biological processes and has been found in many species. Here, the first complete structure of the rabbit HINT1-adenosine complex is reported at 1.10 A resolution, which is one of the highest resolutions obtained for a HINT1 structure. The final structure has an R(cryst) of 14.25% (R(free) = 16.77%) and the model exhibits good stereochemical qualities. A detailed analysis of the atomic resolution data allowed an update of the details of the protein structure in comparison to previously published data.

• Kwon MA, Kim HS, Yang TH, Song BK, Song JK
• High-level expression and characterization of Fusarium solani cutinase in Pichia pastoris.
• Protein Expr Purif. 2009; 68: 104-9
• Display abstract

• High-level extracellular production of Fusarium solani cutinase was achieved using a Pichia pastoris expression system. The cutinase-encoding gene was cloned into pPICZalphaA with the Saccharomyces cerevisiae alpha-factor signal sequence and methanol-inducible alcohol oxidase promoter by two different ways. The additional sequences of the c-myc epitope and (His)6-tag of the vector were fused to the C-terminus of cutinase, while the other expression vector was constructed without any additional sequence. P. pastoris expressing the non-tagged cutinase exhibited about two- and threefold higher values of protein amount and cutinase activity in the culture supernatant, respectively. After simple purification by diafiltration process, both cutinases were much the same in the specific activity and the biochemical properties such as the substrate specificity and the effects of temperature and pH. In conclusion, the high-level secretion of F. solani cutinase in P. pastoris was demonstrated for the first time and would be a promising alternative to many expression systems previously used for the large-scale production of F. solani cutinase in Saccharomyces cerevisiae as well as Escherichia coli.

• Matak MY, Moghaddam ME
• The role of short-range Cys171-Cys178 disulfide bond in maintaining cutinase active site integrity: a molecular dynamics simulation.
• Biochem Biophys Res Commun. 2009; 390: 201-4
• Display abstract

• Understanding structural determinants in enzyme active site integrity can provide a good knowledge to design efficient novel catalytic machineries. Fusarium solani pisi cutinase with classic triad Ser-His-Asp is a promising enzyme to scrutinize these structural determinants. We performed two MD simulations: one, with the native structure, and the other with the broken Cys171-Cys178 disulfide bond. This disulfide bond stabilizes a turn in active site on which catalytic Asp175 is located. Functionally important H-bonds and atomic fluctuations in catalytic pocket have been changed. We proposed that this disulfide bond within active site can be considered as an important determinant of cutinase active site structural integrity.

• Liu Z et al.
• Structural and functional studies of Aspergillus oryzae cutinase: enhanced thermostability and hydrolytic activity of synthetic ester and polyester degradation.
• J Am Chem Soc. 2009; 131: 15711-6
• Display abstract

• Cutinases are responsible for hydrolysis of the protective cutin lipid polyester matrix in plants and thus have been exploited for hydrolysis of small molecule esters and polyesters. Here we explore the reactivity, stability, and structure of Aspergillus oryzae cutinase and compare it to the well-studied enzyme from Fusarium solani. Two critical differences are highlighted in the crystallographic analysis of the A. oryzae structure: (i) an additional disulfide bond and (ii) a topologically favored catalytic triad with a continuous and deep groove. These structural features of A. oryzae cutinase are proposed to result in an improved hydrolytic activity and altered substrate specificity profile, enhanced thermostability, and remarkable reactivity toward the degradation of the synthetic polyester polycaprolactone. The results presented here provide insight into engineering new cutinase-inspired biocatalysts with tailor-made properties.

• Rutten L et al.
• Solid-state structural characterization of cutinase-ECE-pincer-metal hybrids.
• Chemistry. 2009; 15: 4270-80
• Display abstract

• The first crystal structures of lipases that have been covalently modified through site-selective inhibition by different organometallic phosphonate-pincer-metal complexes are described. Two ECE-pincer-type d(8)-metal complexes, that is, platinum (1) or palladium (2) with phosphonate esters (ECE = [(EtO)-(O=)P(-O-C(6)H(4)-(NO(2))-4)(-C(3)H(6)-4-(C(6)H(2)-(CH(2)E)(2))](-); E = NMe(2) or SMe) were introduced prior to crystallization and have been shown to bind selectively to the Ser(120) residue in the active site of the lipase cutinase to give cut-1 (platinum) or cut-2 (palladium) hybrids. For all five presented crystal structures, the ECE-pincer-platinum or -palladium head group sticks out of the cutinase molecule and is exposed to the solvent. Depending on the nature of the ECE-pincer-metal head group, the ECE-pincer-platinum and -palladium guests occupy different pockets in the active site of cutinase, with concomitant different stereochemistries on the phosphorous atom for the cut-1 (S(P)) and cut-2 (R(P)) structures. When cut-1 was crystallized under halide-poor conditions, a novel metal-induced dimeric structure was formed between two cutinase-bound pincer-platinum head groups, which are interconnected through a single mu-Cl bridge. This halide-bridged metal dimer shows that coordination chemistry is possible with protein-modified pincer-metal complexes. Furthermore, we could use NCN-pincer-platinum complex 1 as site-selective tool for the phasing of raw protein diffraction data, which shows the potential use of pincer-platinum complex 1 as a heavy-atom derivative in protein crystallography.

• Vidinha P, Augusto V, Nunes J, Lima JC, Cabral JM, Barreiros S
• Probing the microenvironment of sol-gel entrapped cutinase: the role of added zeolite NaY.
• J Biotechnol. 2008; 135: 181-9
• Display abstract

• Cutinase, an esterase from Fusarium solani pisi, was immobilized in sol-gel matrices of composition 1:5 tetramethoxysilane (TMOS):n-alkyltrimethoxysilane (n-alkylTMS). Fluorescence spectroscopy using the single tryptophan (Trp-69) residue of cutinase as a probe revealed that the polarity of the matrices decreased as their hydrophobicity increased up to the TMOS/n-butylTMS pair, which correlates with an increase in cutinase activity. Fluorescence emission was suppressed (a higher than two orders of magnitude reduction) in the TMOS/n-octylTMS matrix, suggesting a greater proximity of the tryptophan to a nearby disulfide bridge. When sol-gel matrices were prepared with added zeolite NaY, the fluorescence emission intensity maximum (lambda(max)) of the tryptophan did not change. And although the presence of the zeolite led to the recovery of fluorescence emission from the TMOS/n-octylTMS matrix, the corresponding lambda(max) fell in line with the values obtained for the matrices with lower n-alkyl chain lengths, indicating that the tryptophan does not sense the zeolite. On the other hand, the presence of the zeolite led to increases in cutinase activity in all the matrices. This suggests that the zeolite is in a position to affect the active site of the enzyme, located at the opposite pole of the enzyme molecule. Scanning electron microscopy and energy dispersive X-ray spectroscopy revealed that the zeolite particles were segregated to the pores of the matrices. Optical microscopy following the staining of the protein with a fluorescent dye showed that the enzyme was distributed throughout the material, and tended to accumulate around zeolite particles. By promoting the accumulation of the enzyme at the pores of the material, the zeolite should improve the accessibility of the enzyme to the substrates and lead to a higher enzymatic activity. Data obtained for sol-gel matrices with epoxy or SH groups provided further evidence that cutinase responded to changes in the chemical nature of the precursors.

• Sigala PA et al.
• Testing geometrical discrimination within an enzyme active site: constrained hydrogen bonding in the ketosteroid isomerase oxyanion hole.
• J Am Chem Soc. 2008; 130: 13696-708
• Display abstract

• Enzymes are classically proposed to accelerate reactions by binding substrates within active-site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1-1 A scale of most substrate rearrangements. The flexibility of active-site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together 1.2-1.5 A resolution X-ray crystallography, (1)H and (19)F NMR spectroscopy, quantum mechanical calculations, and transition-state analogue binding measurements to test the distance scale on which noncovalent forces can constrain the structural relaxation or translation of side chains and ligands along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side-chain reorientation and prevent hydrogen bond shortening by 0.1 A or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguishable in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function.

• Micaelo NM, Soares CM
• Protein structure and dynamics in ionic liquids. Insights from molecular dynamics simulation studies.
• J Phys Chem B. 2008; 112: 2566-72
• Display abstract

• We present in this work the first molecular simulation study of an enzyme, the serine protease cutinase from Fusarium solani pisi, in two ionic liquids (ILs): 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) and 1-butyl-3-methylimidazolium nitrate ([BMIM][NO(3)]). We tested different water contents in these ILs at room temperature (298 K) and high temperature (343 K), and we observe that the enzyme structure is highly dependent on the amount of water present in the IL media. We show that the enzyme is preferentially stabilized in [BMIM][PF6] at 5-10% (w/w) (weight of water over protein) water content at room temperature. [BMIM][PF6] renders a more nativelike enzyme structure at the same water content of 5-10% (w/w) as previously found for hexane, and the system displays a similar bell-shape-like dependence with the water content in the IL media. [BMIM][PF6] is shown to increase significantly the protein thermostability at high temperatures, especially at low hydration. Our analysis indicates that the enzyme is less stabilized in [BMIM][NO(3)] relative to [BMIM][PF6] at both temperatures, most likely due to the strong affinity of the [NO(3)]- anion toward the protein main chain. These findings are in accordance with the experimental knowledge for these two ionic liquids. We also show that these ILs "strip off" most of the water from the enzyme surface in a degree similar to that found for polar organic solvents such as acetonitrile, and that the remaining waters at the enzyme surface are organized in many small clusters.

• Kondrashov DA, Van Wynsberghe AW, Bannen RM, Cui Q, Phillips GN Jr
• Protein structural variation in computational models and crystallographic data.
• Structure. 2007; 15: 169-77
• Display abstract

• Normal mode analysis offers an efficient way of modeling the conformational flexibility of protein structures. We use anisotropic displacement parameters from crystallography to test the quality of prediction of both the magnitude and directionality of conformational flexibility. Normal modes from four simple elastic network model potentials and from the CHARMM force field are calculated for a data set of 83 diverse, ultrahigh-resolution crystal structures. While all five potentials provide good predictions of the magnitude of flexibility, all-atom potentials have a clear edge at prediction of directionality, and the CHARMM potential has the highest prediction quality. The low-frequency modes from different potentials are similar, but those computed from the CHARMM potential show the greatest difference from the elastic network models. The comprehensive evaluation demonstrates the costs and benefits of using normal mode potentials of varying complexity.

• Sorensen JD, Petersen EI, Wiebe MG
• Production of Fusarium solani f. sp. pisi cutinase in Fusarium venenatum A3/5.
• Biotechnol Lett. 2007; 29: 1227-32
• Display abstract

• Fusarium venenatum A3/5 was transformed using the Aspergillus niger expression plasmid, pIGF, in which the coding sequence for the F. solani f. sp. pisi cutinase gene had been inserted in frame, with a KEX2 cleavage site, with the truncated A. niger glucoamylase gene under control of the A. niger glucoamylase promoter. The transformant produced up to 21 U cutinase l(-1) in minimal medium containing glucose or starch as the primary carbon source. Glucoamylase (165 U l(-1) or 8 mg l(-1)) was also produced. Both the transformant and the parent strain produced cutinase in medium containing cutin.

• Eyal E, Chennubhotla C, Yang LW, Bahar I
• Anisotropic fluctuations of amino acids in protein structures: insights from X-ray crystallography and elastic network models.
• Bioinformatics. 2007; 23: 17584-17584
• Display abstract

• MOTIVATION: A common practice in X-ray crystallographic structure refinement has been to model atomic displacements or thermal fluctuations as isotropic motions. Recent high-resolution data reveal, however, significant departures from isotropy, described by anisotropic displacement parameters (ADPs) modeled for individual atoms. Yet, ADPs are currently reported for a limited set of structures, only. RESULTS: We present a comparative analysis of the experimentally reported ADPs and those theoretically predicted by the anisotropic network model (ANM) for a representative set of structures. The relative sizes of fluctuations along different directions are shown to agree well between experiments and theory, while the cross-correlations between the (x-, y- and z-) components of the fluctuations show considerable deviations. Secondary structure elements and protein cores exhibit more robust anisotropic characteristics compared to disordered or flexible regions. The deviations between experimental and theoretical data are comparable to those between sets of experimental ADPs reported for the same protein in different crystal forms. These results draw attention to the effects of crystal form and refinement procedure on experimental ADPs and highlight the potential utility of ANM calculations for consolidating experimental data or assessing ADPs in the absence of experimental data. AVAILABILITY: The ANM server at http://www.ccbb.pitt.edu/anm is upgraded to permit users to compute and visualize the theoretical ADPs for any PDB structure, thus providing insights into the anisotropic motions intrinsically preferred by equilibrium structures. SUPPLEMENTARY INFORMATION: Two Supplementary Material files can be accessed at the journal website. The first presents the tabulated results from computations (Pearson correlations and KL distances with respect to experimental ADPs) reported for each of the 93 proteins in Set I (the averages over all proteins are presented above in Table 3). The second file consists of three sections: (A) detailed derivation of Equation (7), (B) analysis of the effect of ANM parameters on computed ADPs and identification of parameters that achieve optimal correlation with experiments and (C) description of the method for computing the tangential and radial components of equilibrium fluctuations.

• Schmidt A, Lamzin VS
• Extraction of functional motion in trypsin crystal structures.
• Acta Crystallogr D Biol Crystallogr. 2005; 61: 1132-9
• Display abstract

• The analysis of anisotropic atomic displacement parameters for the direct extraction of functionally relevant motion from X-ray crystal structures of Fusarium oxysporum trypsin is presented. Several atomic resolution structures complexed with inhibitors or substrates and determined at different pH values and temperatures were investigated. The analysis revealed a breathing-like molecular motion conserved across trypsin structures from two organisms and three different crystal forms. Directional motion was observed suggesting a change of the width of the substrate-binding cleft and a change in the length of the specificity pocket. The differences in direction of motion across the structures are dependent on the mode of substrate or inhibitor binding and the chemical environment around the active-site residues. Together with the occurrence of multiple-residue conformers, they reflect spatial rearrangement throughout the deacylation pathway.

• Micaelo NM, Teixeira VH, Baptista AM, Soares CM
• Water dependent properties of cutinase in nonaqueous solvents: a computational study of enantioselectivity.
• Biophys J. 2005; 89: 999-1008
• Display abstract

• The catalytic properties of enzymes in nonaqueous solvents are known to be dependent on the nature of the solvent. Here we present a molecular modeling study of the enantioselective properties of the enzyme cutinase in hexane under varying hydration conditions. Previous simulation studies have shown that for this model enzyme in hexane, the structural and dynamical properties are affected by the amount of water associated with the protein, being more similar to the aqueous simulation at 5-10% of water content. The implications of the hydration levels on the enzyme resolution of (R,S)-1-phenylethanol and (R,S)-2-phenyl-1-propanol are investigated using free energy calculations of the tetrahedral intermediate (TI) model. With this model system we show that the enzyme enantioselective properties are under the control of the amount of water present in the organic media. Maximum enantioselectivity is achieved at 10% water content. The stabilizing effects of the catalytic histidine on the TI are evaluated at different water contents and shown to be correlated. The correlation between the amount of water present in the media and the structural, dynamical, and thermodynamic properties of the enzyme are examined as well as the active site discriminative power.

• Lou X et al.
• The atomic resolution crystal structure of atratoxin determined by single wavelength anomalous diffraction phasing.
• J Biol Chem. 2004; 279: 39094-104
• Display abstract

• By using single wavelength anomalous diffraction phasing based on the anomalous signal from copper atoms, the crystal structure of atratoxin was determined at the resolution of 1.5 A and was refined to an ultrahigh resolution of 0.87 A. The ultrahigh resolution electron density maps allowed the modeling of 38 amino acid residues in alternate conformations and the location of 322 of 870 possible hydrogen atoms. To get accurate information at the atomic level, atratoxin-b (an analog of atratoxin with reduced toxicity) was also refined to an atomic resolution of 0.92 A. By the sequence and structural comparison of these two atratoxins, Arg(33) and Arg(36) were identified to be critical to their varied toxicity. The effect of copper ions on the distribution of hydrogen atoms in atratoxin was discussed, and the interactions between copper ions and protein residues were analyzed based on a statistical method, revealing a novel pentahedral copper-binding motif.

• Kursula I, Wierenga RK
• Crystal structure of triosephosphate isomerase complexed with 2-phosphoglycolate at 0.83-A resolution.
• J Biol Chem. 2003; 278: 9544-51
• Display abstract

• The atomic resolution structure of Leishmania mexicana triosephosphate isomerase complexed with 2-phosphoglycolate shows that this transition state analogue is bound in two conformations. Also for the side chain of the catalytic glutamate, Glu(167), two conformations are observed. In both conformations, a very short hydrogen bond exists between the carboxylate group of the ligand and the catalytic glutamate. The distance between O11 of PGA and Oepsilon2 of Glu(167) is 2.61 and 2.55 A for the major and minor conformations, respectively. In either conformation, Oepsilon1 of Glu(167) is hydrogen-bonded to a water network connecting the side chain with bulk solvent. This network also occurs in two mutually exclusive arrangements. Despite the structural disorder in the active site, the C termini of the beta strands that construct the active site display the least anisotropy compared with the rest of the protein. The loops following these beta strands display various degrees of anisotropy, with the tip of the dimer interface loop 3 having very low anisotropy and the C-terminal region of the active site loop 6 having the highest anisotropy. The pyrrolidine ring of Pro(168) at the N-terminal region of loop 6 is in a strained planar conformation to facilitate loop opening and product release.

• Lario PI, Sampson N, Vrielink A
• Sub-atomic resolution crystal structure of cholesterol oxidase: what atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity.
• J Mol Biol. 2003; 326: 1635-50
• Display abstract

• The crystal structure of cholesterol oxidase, a 56kDa flavoenzyme was anisotropically refined to 0.95A resolution. The final crystallographic R-factor and R(free) value is 11.0% and 13.2%, respectively. The quality of the electron density maps has enabled modeling of alternate conformations for 83 residues in the enzyme, many of which are located in the active site. The additional observed structural features were not apparent in the previous high-resolution structure (1.5A resolution) and have enabled the identification of a narrow tunnel leading directly to the isoalloxazine portion of the FAD prosthetic group. The hydrophobic nature of this narrow tunnel suggests it is the pathway for molecular oxygen to access the isoalloxazine group for the oxidative half reaction. Resolving the alternate conformations in the active site residues provides a model for the dynamics of substrate binding and a potential oxidation triggered gating mechanism involving access to the hydrophobic tunnel. This structure reveals that the NE2 atom of the active site histidine residue, H447, critical to the redox activity of this flavin oxidase, acts as a hydrogen bond donor rather than as hydrogen acceptor. The atomic resolution structure of cholesterol oxidase has revealed the presence of hydrogen atoms, dynamic aspects of the protein and how side-chain conformations are correlated with novel structural features such as the oxygen tunnel. This new structural information has provided us with the opportunity to re-analyze the roles played by specific residues in the mechanism of the enzyme.

• Shimizu H, Park SY, Shiro Y, Adachi Si
• X-ray structure of nitric oxide reductase (cytochrome P450nor) at atomic resolution.
• Acta Crystallogr D Biol Crystallogr. 2002; 58: 81-9
• Display abstract

• Crystal structures of the nitric oxide reductase cytochrome P450nor (P450nor) in the ferric resting and the ferrous carbonmonoxy (CO) states have been determined at 1.00 and 1.05 A resolution, respectively. P450nor consists of 403 amino-acid residues (46 kDa) and is one of the largest proteins refined to this resolution so far. The final models have conventional R factors of 10.2% (ferric resting) and 11.7% (ferrous CO), with mean coordinate errors of 0.028 (ferric resting) and 0.030 A (ferrous CO) as calculated from inversion of the full positional least-squares matrix. Owing to the atomic resolution, novel features are found in the refined structures. Firstly, two orientations of the haem are observed both in the ferric resting and the ferrous CO states. Secondly, a disordered water molecule bound to the haem iron is found in the ferric resting state. In addition, the accurate structures at atomic resolution enabled the examination of general stereochemical parameters that are commonly used in refinement cycles of protein structures.

• Sandalova T, Schneider G, Kack H, Lindqvist Y
• Structure of dethiobiotin synthetase at 0.97 A resolution.
• Acta Crystallogr D Biol Crystallogr. 1999; 55: 610-24
• Display abstract

• The crystal structure of the 224-residue protein dethiobiotin synthetase from Escherichia coli has been refined using X-ray diffraction data at 0.97 A resolution at 100 K. The model, consisting of 4143 protein atoms including 1859 H atoms and 436 solvent sites, was refined to a final R factor of 11.6% for all reflections, and has an estimated mean standard uncertainty for the atomic positions of 0.022 A, derived from inversion of the blocked matrix. The structure was refined with a full anisotropic model for the atomic displacement parameters using SHELX97. Stereochemical restraints were applied throughout the refinement. In the last cycles, the planarity of the peptide bonds was not restrained, resulting in a mean omega value of 179.6 degrees. Analysis of the most anisotropic regions of the protein shows that they form four clusters of residues. Alternate conformations for the side chains of 15 residues and for the main-chain atoms of six residues from three loops were included in the model. An analysis of C-HcO hydrogen bonds shows that such interactions occur rather frequently in DTBS; in total, 16 such hydrogen bonds were found. In the central beta-sheet, 13 C-HcO bonds between carbonyl O and Calpha H atoms were found. Other interactions of this type involve main-chain-side-chain and side-chain-side-chain C-HcO bonds. The model includes 436 water sites, of which 233 molecules form the first hydration shell. Analysis of the protein-solvent interactions shows that about one third of the accessible surface of the enzyme is not covered by ordered solvent. No difference in propensity for ordered solvent close to hydrophilic or hydrophobic surface areas was found. The comparison of the 100 K structure with the structure of the enzyme determined at room temperature shows several regions with different conformation, including areas in the active site, suggesting that structural transitions can occur during flash freezing. This observation questions one of the basic assumptions in the analysis of enzymatic reaction mechanisms using cryocrystallography.

• Harata K, Abe Y, Muraki M
• Crystallographic evaluation of internal motion of human alpha-lactalbumin refined by full-matrix least-squares method.
• J Mol Biol. 1999; 287: 347-58
• Display abstract

• The low temperature form of human alpha-lactalbumin (HAL) was crystallized from a 2H2O solution and its structure was refined to the R value of 0.119 at 1.15 A resolution by the full-matrix least-squares method. Average estimated standard deviations of atomic parameters for non-hydrogen atoms were 0.038 A for coordinates and 0.044 A2 for anisotropic temperature factors (Uij). The magnitude of equivalent isotropic temperature factors (Ueqv) was highly correlated with the distance from the molecular centroid and fitted to a quadratic equation as a function of atomic coordinates. The atomic thermal motion was rather isotropic in the core region and the anisotropy increased towards the molecular surface. The statistical analysis revealed the out-of-plane motion of main-chain oxygen atoms, indicating that peptide groups are in rotational vibration around a Calpha.Calpha axis. The TLS model, which describes the rigid-body motion in terms of translation, libration, and screw motions, was adopted for the evaluation of the molecular motion and the TLS parameters were determined by the least-squares fit to Uij. The reproduced Ueqvcal from the TLS parameters was in fair agreement with observed Ueqv, but differences were found in regions of residues, 5-22, 44-48, 70-75, and 121-123, where Ueqv was larger than Ueqvcal because of large local motions. To evaluate the internal motion of HAL, the contribution of the rigid-body motion was determined to be 42.4 % of Ueqv in magnitude, which was the highest estimation to satisfy the condition that the Uijint tensors of the internal motion have positive eigen values. The internal motion represented with atomic thermal ellipsoids clearly showed local motions different from those observed in chicken-type lysozymes which have a backbone structure very similar to HAL. The result indicates that the internal motion is closely related to biological function of proteins.

• Merritt EA
• Expanding the model: anisotropic displacement parameters in protein structure refinement.
• Acta Crystallogr D Biol Crystallogr. 1999; 55: 1109-17
• Display abstract

• Recent technological improvements in crystallographic data collection have led to a surge in the number of protein structures being determined at atomic or near-atomic resolution. At this resolution, structural models can be expanded to include anisotropic displacement parameters (ADPs) for individual atoms. New protocols and new tools are needed to refine, analyze and validate such models optimally. One such tool, PARVATI, has been used to examine all protein structures (peptide chains >50 residues) for which expanded models including ADPs are available from the Protein Data Bank. The distribution of anisotropy within each of these refined models is broadly similar across the entire set of structures, with a mean anisotropy A in the range 0.4-0.5. This is a significant departure from a purely isotropic model and explains why the inclusion of ADPs yields a substantial improvement in the crystallographic residuals R and Rfree. The observed distribution of anisotropy may prove useful in the validation of very high resolution structures. A more complete understanding of this distribution may also allow the development of improved protein structural models, even at lower resolution.

• Jelsch C, Pichon-Pesme V, Lecomte C, Aubry A
• Transferability of multipole charge-density parameters: application to very high resolution oligopeptide and protein structures.
• Acta Crystallogr D Biol Crystallogr. 1998; 54: 1306-18
• Display abstract

• Crystallography at sub-atomic resolution permits the observation and measurement of the non-spherical character of the electron density (parameterized as multipoles) and of the atomic charges. This fine description of the electron density can be extended to structures of lower resolution by applying the notion of transferability of the charge and multipole parameters. A database of such parameters has been built from charge-density analysis of several peptide crystals. The aim of this study is to assess for which X-ray structures the application of transferability is physically meaningful. The charge-density multipole parameters have been transferred and the X-ray structure of a 310 helix octapeptide Ac-Aib2-L-Lys(Bz)-Aib2-L-Lys(Bz)-Aib2-NHMe refined subsequently, for which diffraction data have been collected to a resolution of 0.82 A at a cryogenic temperature of 100 K. The multipoles transfer resulted in a significant improvement of the crystallographic residual factors wR and wR free. The accumulation of electrons in the covalent bonds and oxygen lone pairs is clearly visible in the deformation electron-density maps at its expected value. The refinement of the charges for nine different atom types led to an additional improvement of the R factor and the refined charges are in good agreement with those of the AMBER molecular modelling dictionary. The use of scattering factors calculated from average results of charge-density work gives a negligible shift of the atomic coordinates in the octapeptide but induces a significant change in the temperature factors (DeltaB approximately 0.4 A2). Under the spherical atom approximation, the temperature factors are biased as they partly model the deformation electron density. The transfer of the multipoles thus improves the physical meaning of the thermal-displacement parameters. The contribution to the diffraction of the different components of the electron density has also been analyzed. This analysis indicates that the electron-density peaks are well defined in the dynamic deformation maps when the thermal motion of the atoms is moderate (B typically lower than 4 A2). In this case, a non-truncated Fourier synthesis of the deformation density requires that the diffraction data are available to a resolution better than 0.9 A.

• Jelsch C, Longhi S, Cambillau C
• Packing forces in nine crystal forms of cutinase.
• Proteins. 1998; 31: 320-33
• Display abstract

• During the characterization of mutants and covalently inhibited complexes of Fusarium solani cutinase, nine different crystal forms have been obtained so far. Protein mutants with a different surface charge distribution form new intermolecular salt bridges or long-range electrostatic interactions that are accompanied by a change in the crystal packing. The whole protein surface is involved in the packing contacts and the hydrophobicities of the protein surfaces in mutual contact turned out to be noncorrelated, which indicates that the packing interactions are nonspecific. In the case of the hydrophobic variants, the packing contacts showed some specificity, as the protein in the crystal tends to form either crystallographic or noncrystallographic dimers, which shield the hydrophobic surface from the solvent. The likelihood of surface atoms to be involved in a crystal contact is the same for both polar and nonpolar atoms. However, when taking areas in the 200-600 A2 range, instead of individual atoms, the either highly hydrophobic or highly polar surface regions were found to have an increased probability of establishing crystal lattice contacts. The protein surface surrounding the active-site crevice of cutinase constitutes a large hydrophobic area that is involved in packing contacts in all the various crystalline contexts.

• Merritt EA, Kuhn P, Sarfaty S, Erbe JL, Holmes RK, Hol WG
• The 1.25 A resolution refinement of the cholera toxin B-pentamer: evidence of peptide backbone strain at the receptor-binding site.
• J Mol Biol. 1998; 282: 1043-59
• Display abstract

• Crystals of the 61 kDa complex of the cholera toxin B-pentamer with the ganglioside GM1 receptor pentasaccharide diffract to near-atomic resolution. We have refined the crystallographic model for this complex using anisotropic displacement parameters for all atoms to a conventional crystallographic residual R=0.129 for all observed Bragg reflections in the resolution range 22 A to 1.25 A. Remarkably few residues show evidence of discrete conformational disorder. A notable exception is a minority conformation found for the Cys9 side-chain, which implies that the Cys9-Cys86 disulfide linkage is incompletely formed. In all five crystallographically independent instances, the peptide backbone in the region of the receptor-binding site shows evidence of strain, including unusual bond lengths and angles, and a highly non-planar (omega=153.7(7) degrees) peptide group between residues Gln49 and Val50. The location of well-ordered water molecules at the protein surface is notable reproduced among the five crystallographically independent copies of the peptide chain, both at the receptor-binding site and elsewhere. The 5-fold non-crystallographic symmetry of this complex allows an evaluation of the accuracy, reproducibility, and derived error estimates from refinement of large structures at near-atomic resolution. We find that blocked-matrix treatment of parameter covariance underestimates the uncertainty of atomic positions in the final model by approximately 10% relative to estimates based either on full-matrix inversion or on the 5-fold non-crystallographic symmetry.

• Harata K, Abe Y, Muraki M
• Full-matrix least-squares refinement of lysozymes and analysis of anisotropic thermal motion.
• Proteins. 1998; 30: 232-43
• Display abstract

• Crystal structures of turkey egg lysozyme (TEL) and human lysozyme (HL) were refined by full-matrix least-squares method using anisotropic temperature factors. The refinement converged at the conventional R-values of 0.104 (TEL) and 0.115 (HL) for reflections with Fo > 0 to the resolution of 1.12 A and 1.15 A, respectively. The estimated r.m.s. coordinate errors for protein atoms were 0.031 A (TEL) and 0.034 A (HL). The introduction of anisotropic temperature factors markedly reduced the R-value but did not significantly affect the main chain coordinates. The degree of anisotropy of atomic thermal motion has strong positive correlation with the square of distance from the molecular centroid. The ratio of the radial component of thermal ellipsoid to the r.m.s. magnitude of three principal components has negative correlation with the distance from the molecular centroid, suggesting the domination of libration rather than breathing motion. The TLS model was applied to elucidate the characteristics of the rigid-body motion. The TLS tensors were determined by the least-squares fit to observed temperature factors. The profile of the magnitude of reproduced temperature factors by the TLS method well fitted to that of observed B(eqv). However, considerable disagreement was observed in the shape and orientation of thermal ellipsoid for atoms with large temperature factors, indicating the large contribution of local motion. The upper estimate of the external motion, 67% (TEL) and 61% (HL) of B(eqv), was deduced from the plot of the magnitude of TLS tensors determined for main chain atoms which were grouped into shells according to the distance from the center of libration. In the external motion, the translational portion is predominant and the contribution of libration and screw motion is relatively small. The internal motion, estimated by subtracting the upper estimate of the external motion from the observed temperature factor, is very similar between TEL and HL in spite of the difference in 54 of 130 amino acid residues and in crystal packing, being suggested to reflect the intrinsic internal motion of chicken-type lysozymes.

• Gregoriou M et al.
• The structure of a glycogen phosphorylase glucopyranose spirohydantoin complex at 1.8 A resolution and 100 K: the role of the water structure and its contribution to binding.
• Protein Sci. 1998; 7: 915-27
• Display abstract

• A glucopyranose spirohydantoin (a pyranose analogue of the potent herbicide, hydantocidin) has been identified as the highest affinity glucose analogue inhibitor of glycogen phosphorylase b (GPb). In order to elucidate the structural features that contribute to the binding, the structures of GPb in the native T state conformation and in complex with glucopyranose spirohydantoin have been determined at 100 K to 2.0 A and 1.8 A resolution, respectively, and refined to crystallographic R values of 0.197 (R[free] 0.248) and 0.182 (R[free] 0.229), respectively. The low temperature structure of GPb is almost identical to that of the previously determined room temperature structure, apart from a decrease in overall atomic temperature factors ((B) room temperature GPb = 34.9 A2; (B) 100 K GPb = 23.4 A2). The glucopyranose spirohydantoin inhibitor (Ki = 3.0 microM) binds at the catalytic site and induces small changes in two key regions of the protein: the 280s loop (residues 281-286) that results in a decrease in mobility of this region, and the 380s loop (residues 377-385) that undergoes more significant shifts in order to optimize contact to the ligand. The hydantoin group, that is responsible for increasing the affinity of the glucose compound by a factor of 10(3), makes only one hydrogen bond to the protein, from one of its NH groups to the main chain oxygen of His377. The other polar groups of the hydantoin group form hydrogen bonds to five water molecules. These waters are involved in extensive networks of hydrogen bonds and appear to be an integral part of the protein structure. Analysis of the water structure at the catalytic site of the native enzyme, shows that five waters are displaced by ligand binding and that there is a significant decrease in mobility of the remaining waters on formation of the GPb-hydantoin complex. The ability of the inhibitor to exploit existing waters, to displace waters and to recruit new waters appears to be important for the high affinity of the inhibitor.

• Nicolas A et al.
• Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state.
• Biochemistry. 1996; 35: 398-410
• Display abstract

• Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.

• Mannesse ML et al.
• Cutinase from Fusarium solani pisi hydrolyzing triglyceride analogues. Effect of acyl chain length and position in the substrate molecule on activity and enantioselectivity.
• Biochemistry. 1995; 34: 6400-7
• Display abstract

• Triglyceride analogues were synthesized in which one of the primary acyl ester functions has been replaced by an alkyl group and the secondary acyl ester bond has been replaced by an acyl amino bond. The chain length at either position was varied, and both (R)- and (S)-enantiomers of each compound were synthesized. These pseudo triglycerides contain only one hydrolyzable ester bond, and they are ideally suited to studying the influence of the chain length at the 1-, 2-, and 3-position on lipase activity and on stereopreference. These substrates were used to characterize cutinase from Fusarium solani pisi. Our results show that the activity of cutinase is very sensitive to the length and distribution of the acyl chains and that the highest activities are found when the chains at positions 1 and 3 contain three or four carbon atoms. The enzyme preferentially hydrolyzes the (R)-enantiomers, but this preference is strongly dependent on the acyl chain length distribution, with (R) over (S) activity ratios varying from about 30 to 1. This enantioselectivity was found in three different assay systems: a mixed micellar, a reverse micellar, and a monolayer study. Our data suggest that at least two alkyl chains of the pseudo triglycerides must be fixed during hydrolysis. Therefore, these substrates were used to characterize mutants of cutinase with mutations in putative lipid binding domains. Two mutants (A85F and A85W) have increased activities. The results obtained with these mutants suggest an interaction of the acyl chain of the scissile ester bond with a surface loop, comprising residues 80-90, in the enzyme-substrate complex.

• Fields BA, Bartsch HH, Bartunik HD, Cordes F, Guss JM, Freeman HC
• Accuracy and precision in protein crystal structure analysis: two independent refinements of the structure of poplar plastocyanin at 173 K.
• Acta Crystallogr D Biol Crystallogr. 1994; 50: 709-30
• Display abstract

• The structure of the copper protein plastocyanin from poplar leaves (Populus nigra var. italica) at 173 K has been subjected to two independent refinements, using a single set of synchrotron X-ray data at 1.6 A resolution. Energy-restrained refinement using the program EREF resulted in lower root-mean-square deviations from ideal geometry (e.g. 0.011 A for bond lengths) but a higher residual R (0.153) than restrained least-squares refinement using the program PROLSQ (0.014 A, 0.132). Electron-density difference maps in both refinements provided evidence for disorder at some side chains and solvent atoms, and the PROLSQ refinement made allowance for this disorder. The number of solvent sites identified at the 4sigma(rho) level was 171 in the EREF refinement and 189 in the PROLSQ refinement; 159 of the solvent sites are common to both refinements within 1 A. The root-mean-square differences between the atomic positions produced by the two refinements are 0.08 A for C(alpha) atoms, 0.08 A for backbone atoms and 0.12 A for all non-H atoms (excluding six obvious outliers) of the protein molecule. The two sets of Cu-ligand bond lengths differ by up to 0.07 A, and the ligand-Cu-ligand angles by up to 7 degrees. At 173 K the volume of the unit cell is 4.2% smaller than at 295 K. Greater order in the solvent region is indicated by the location of 79 more solvent sites, the identification of extensive networks of hydrogen-bonded rings of solvent molecules, and a general decrease in the thermal parameters. Within the unit cell, the protein molecules are significantly translated and rotated from their positions at ambient temperature. An important structural change at low temperature is a 180 degrees flip of the peptide group at Ser48-Gly49. Nearly all other significant differences between the structures of the protein at 173 and 295 K occur at exposed side chains. If the backbone atoms in the 173 and 295 K structures are superposed, excluding atoms involved in the peptide flip, the root-mean- square difference between the positions of 393 atoms is 0.25 A. Two internal water molecules, not included in previous descriptions of poplar plastocyanin, have been located. The plastocyanin Cu-site geometry at 173 K is not significantly different from that at 295 K. If plastocyanin undergoes a change in Cu-site geometry at low temperature, as has been suggested on the basis of resonance Raman spectroscopic evidence, then the change is not detected within the limits of precision of the present results.

• Ringe D, Petsko GA
• Study of protein dynamics by X-ray diffraction.
• Methods Enzymol. 1986; 131: 389-433
• Display abstract

• Properly carried out, high-resolution X-ray diffraction data collection followed by careful least-squares refinement can give the spatial distribution of the high-frequency mean-square displacements in a protein. These displacements reflect both individual atomic fluctuations in hard variables (bond lengths and bond angles) and collective motions involving soft variables (torsion angles, nonbonded interactions). Lower frequency, large amplitude motions and rapid but improbable motions are not quantifiable, but they may lead to such complete disorder that their existence can at least be inferred from the absence of interpretable electron density for some sections of the structure. Interior residues are more rigid than groups on the surface, and structural constraints are reflected in restricted motion even for surface residues. Amplitudes of motion of 0.5 A or greater are not uncommon. The temperature dependence of these fast motions varies considerably over the structure. In general, large [chi 2] values have large temperature dependence, while small displacements are less affected by temperature; however, exceptions are common. Significant reduction in [chi 2] on cooling establishes that proteins are mobile even in the crystalline state, and that static disorder is not the dominant contributor to the individual mean square displacements. Disordered regions in electron density maps are no longer automatically taken as signs of errors in structure determination. It is now recognized that the absence of strong electron density is often an indicator of conformational flexibility. Some of the functional roles for protein dynamics are beginning to be understood. Missing from these results are the physicochemical details that can be extracted from thermal motion analysis of small molecule crystal structures. Application of these methods to protein data is very difficult, but it is well to remember that just over 10 years ago it was commonly felt that protein structures could not even be refined. Certainly some small, well-diffracting proteins should be amenable to many of the sophisticated small-molecule analyses, as they yield X-ray data to resolutions comparable to simple organic structures. The most important type of analysis that awaits is anisotropic B factor refinement, which would give the principal directions of motion added to the amplitude information now obtained. Unfortunately, refinement of unrestrained anisotropic thermal elipsoids requires six parameters for each atom instead of a single isotropic B parameter, and even 1.5 A resolution data do not provide enough overdeterminacy.(ABSTRACT TRUNCATED AT 400 WORDS)

• Lin TS, Kolattukudy PE
• Structural studies on cutinase, a glycoprotein containing novel amino acids and glucuronic acid amide at the N terminus.
• Eur J Biochem. 1980; 106: 341-51
• Display abstract

• Cutinase I and cutinase II, two extracellular enzymes produced by Fusarium solani pisi, were shown to be glycoproteins containing 4.3% and 5.1% carbohydrates, respectively. Upon treatment with alkali both enzymes generated chromophores which absorbed at 241 nm. Treatment of both proteins with alkaline NaB3H4 gave labeled protein and labeled monosaccharides. Hydrolysis of the labeled protein followed by chromatographic and enzymatic analyses of the products showed that alanine, 2-aminobutyrate, phenylalanine, tyrosine and L-gulonic acid accounted for nearly all of the 3H contained in the protein. The four labeled amino acids were shown to be 1:1 mixture of D and L isomers and 3H was nearly equally distributed between alpha and beta positions in each amino acid. The N-terminal amino group of cutinase I did not react with either phenylisothiocyanate of dansyl chloride. This amino group was suggested to be in amide linkage with glucuronic acid because upon treatment of the protein with neutral NaB3H4, gulonic acid attached to the protein became labeled and only gulonic acid was labeled when the protein was deglycosylated with HF prior to alkaline NaB3H4 treatment. Furthermore, N-gulonyglycine was isolated from the pronase digest of the labeled protein. Chromatographic identification and quantification of the labeled carbohydrates released from cutinase I by alkaline NaB3H4 showed that one mole of cutinase I has one mole each of mannose, arabinose, N-acetylglucosamine, and glucuronic acid O-glycosidically linked to serine, threonine, beta-hydroxyphenylalanine, and beta-hydroxytyrosine. In addition, the N-terminal glycine is in amide linkage with glucuronic acid. Since almost identical experimental results were obtained with cutinase II this protein is also suggested to have the same structural features as those suggested above for cutinase I.

• Lin TS, Kolattukudy PE
• Direct evidence for the presence of beta-hydroxyphenylalanine and beta-hydroxytyrosine in cutinase from Fusarium solani pisi.
• Arch Biochem Biophys. 1979; 196: 255-64