Protein packing: dependence on protein size, secondary structure and amino acid composition

We have used the occluded surface algorithm to estimate the packing of both buried and exposed amino acid residues in protein structures. This method works equally well for buried residues and solvent-exposed residues in contrast to the commonly used Voronoi method that works directly only on buried residues. The atomic packing of individual globular proteins may vary significantly from the average packing of a large data set of globular proteins. Here, we demonstrate that these variations in protein packing are due to a complex combination of protein size, secondary structure composition and amino acid composition. Differences in protein packing are conserved in protein families of similar structure despite significant sequence differences. This conclusion indicates that quality assessments of packing in protein structures should include a consideration of various parameters including the packing of known homologous proteins. Also, modeling of protein structures based on homologous templates should take into account the packing of the template protein structure.     

Using physical features of protein core packing to distinguish real proteins from decoys

The ability to consistently distinguish real protein structures from computationally generated model decoys is not yet a solved problem. One route to distinguish real protein structures from decoys is to delineate the important physical features that specify a real protein. For example, it has long been appreciated that the hydrophobic cores of proteins contribute significantly to their stability. We used two sources to obtain datasets of decoys to compare with real protein structures: submissions to the biennial Critical Assessment of protein Structure Prediction competition, in which researchers attempt to predict the structure of a protein only knowing its amino acid sequence, and also decoys generated by 3DRobot, which have user‐specified global root‐mean‐squared deviations from experimentally determined structures. Our analysis revealed that both sets of decoys possess cores that do not recapitulate the key features that define real protein cores. In particular, the model structures appear more densely packed (because of energetically unfavorable atomic overlaps), contain too few residues in the core, and have improper distributions of hydrophobic residues throughout the structure. Based on these observations, we developed a feed‐forward neural network, which incorporates key physical features of protein cores, to predict how well a computational model recapitulates the real protein structure without knowledge of the structure of the target sequence. By identifying the important features of protein structure, our method is able to rank decoy structures with similar accuracy to that obtained by state‐of‐the‐art methods that incorporate many additional features. The small number of physical features makes our model interpretable, emphasizing the importance of protein packing and hydrophobicity in protein structure prediction.     

Crystallographic analysis of an "anticalin" with tailored specificity for fluorescein reveals high structural plasticity of the lipocalin loop region

The artificial lipocalin FluA with novel specificity toward fluorescein was derived via combinatorial engineering from the bilin-binding protein, BBP by exchange of 16 amino acids in the ligand pocket. Here, we describe the crystal structure of FluA at 2.0 A resolution in the space group P2(1) with two protein-ligand complexes in the asymmetric unit. In both molecules, the characteristic beta-barrel architecture with the attached alpha-helix is well preserved. In contrast, the four loops at one end of the beta-barrel that form the entrance to the binding site exhibit large conformational deviations from the wild-type protein, which can be attributed to the sidechain replacements. Specificity for the new ligand is furnished by hydrophobic packing, charged sidechain environment, and hydrogen bonds with its hydroxyl groups. Unexpectedly, fluorescein is bound in a much deeper cavity than biliverdin IX(gamma) in the natural lipocalin. Triggered by the substituted residues, unmutated sidechains at the bottom of the binding site adopt conformations that are quite different from those observed in the BBP, illustrating that not only the loop region but also the hydrophobic interior of the beta-barrel can be reshaped for molecular recognition. Particularly, Trp 129 participates in a tight stacking interaction with the xanthenolone moiety, which may explain the ultrafast electron transfer that occurs on light excitation of the bound fluorescein. These structural findings support our concept of using lipocalins as a scaffold for the engineering of so-called "anticalins" directed against prescribed targets as an alternative to recombinant antibody fragments.     

RosettaHoles2: A volumetric packing measure for protein structure refinement and validation

We present an improved version of RosettaHoles, a methodology for quantitative and visual characterization of protein core packing. RosettaHoles2 features a packing measure more rapidly computable, accurate and physically transparent, as well as a new validation score intended for structures submitted to the Protein Data Bank. The differential packing measure is parameterized to maximize the gap between computationally generated and experimentally determined X‐ray structures, and can be used in refinement of protein structure models. The parameters of the model provide insight into components missing in current force fields, and the validation score gives an upper bound on the X‐ray resolution of Protein Data Bank structures; a crystal structure should have a validation score as good as or better than its resolution.     

High resolution crystal structures of the Escherichia coli lytic transglycosylase Slt70 and its complex with a peptidoglycan fragment.

The 70 kDa soluble lytic transglycosylase (Slt70) from Escherichia coli is an exo-muramidase, that catalyses the cleavage of the glycosidic bonds between N -acetylmuramic acid and N -acetylglucosamine residues in peptidoglycan, the main structural component of the bacterial cell wall. This cleavage is accompanied by the formation of a 1,6-anhydro bond between the C1 and O6 atoms in the N -acetylmuramic acid residue (anhMurNAc). Crystallographic studies at medium resolution revealed that Slt70 is a multi-domain protein consisting of a large ring-shaped alpha-superhelix with on top a catalytic domain, which resembles the fold of goose-type lysozyme. Here we report the crystal structures of native Slt70 and of its complex with a 1,6-anhydromuropeptide solved at nominal resolutions of 1.65 A and 1.90 A, respectively. The high resolution native structure reveals the details on the hydrogen bonds, electrostatic and hydrophobic interactions that stabilise the catalytic domain and the alpha-superhelix. The building-block of the alpha-superhelix is an "up-down-up-down" four-alpha-helix bundle involving both parallel and antiparallel helix pairs. Stabilisation of the fold is provided through an extensive packing of apolar atoms, mostly from leucine and alanine residues. It lacks, however, an internal consensus sequence that characterises other super-secondary helical folds like the beta-helix in pectate lyase or the (beta-alpha)-helix in the ribonuclease inhibitor. The 1, 6-anhydromuropeptide product binds in a shallow groove adjacent to the peptidoglycan-binding groove of the catalytic domain. The groove is formed by conserved residues at the interface of the catalytic domain and the alpha-superhelix. The structure of the Slt70-1, 6-anhydromuropeptide complex confirms the presence of a specific binding-site for the peptide moieties of the peptidoglycan and it substantiates the notion that Slt70 starts the cleavage reaction at the anhMurNAc end of the peptidoglycan.     

Comparison of the predicted structure for the activated form of the P21 protein with the X-ray crystal structure

The predicted conformation and position of the central transforming region (residues 55–67) of the p21 protein are compared with the conformation and position of this segment in a recently determined X-ray crystal structure of residues 1–166 of this protein in the activated state bound to a nonhydrolyzable GTP derivative. We previously predicted that this segment of the protein would adopt a roughly extended conformation from Ile 55-Thr 58, a reverse turn at Ala 59-Gln 61, followed by an -helix from Glu 62-Met 67. We further predicted that this region of the activated protein occupies a position that is virtually identical to corresponding regions in the homologous purine nucleotide-binding proteins, bacterial elongation factor (EF-tu), and adenylate kinase (ADK). We find that there is a close correspondence between the conformation and position of our predicted structure and those found in the X-ray crystal structure. A mechanism for activation of the protein is proposed and is corroborated by X-ray crystallographic data.     

Yeast iso-1-cytochrome c. A 2.8 A resolution three-dimensional structure determination

A molecular replacement approach, augmented with the results of predictive modeling procedures, solvent accessibility studies, packing analyses and translational coefficient searches, has been used to elucidate the 2.8 A (1 A = 0.1 nm) resolution structure of yeast iso-1-cytochrome c. An examination of the polypeptide chain folding of this protein shows it to have unique conformations in three regions, upon comparison with the structures of other eukaryotic cytochromes c. These include: residues -5 to +1 at the N-terminal end of the polypeptide chain, which are in an extended conformation and project in large part off the surface of the protein; residues 19 to 26, which form a surface beta-loop on the His18 ligand side of the central heme group; and, the C-terminal end of the helical segment composed of residues 49 to 56, which serves to form a part of the heme pocket. Structural studies also show that the highly reactive sulfhydryl group of Cys102 is buried within a hydrophobic region in the monomer form of yeast iso-1-cytochrome c. Dimerization of yeast iso-1-cytochrome c through disulfide bond formation between two such residues would require a substantial conformational change in the C-terminal helix of this protein. Another unique structural feature, the trimethylated side-chain of Lys72, is located on the surface of yeast iso-1-cytochrome c near the solvent-exposed edge of the bound heme prosthetic group. On the basis of the results of these and other structural studies, an analysis of the spatial conservation of structural features in the heme pocket of eukaryotic cytochromes c has been conducted. It was found that the residues involved could be divided into three general classes. The current structural analyses and additional modeling studies have also been used to explain the altered functional properties observed for mutant yeast iso-1-cytochrome c proteins.     

Structure of azurin from Alcaligenes denitrificans refinement at 1.8 A resolution and comparison of the two crystallographically independent molecules

The structure of the blue copper protein azurin, from Alcaligenes denitrificans, has been refined crystallographically by restrained least-squares methods. The final crystallographic R value for 21,980 observed reflections to 1.8 A (1 A = 0.1 nm) resolution is 0.157. The asymmetric unit of the crystal contains two independent azurin molecules, the model for which comprises 1973 protein atoms, together with three SO2-4 ions, and 281 water molecules. Comparison of the two molecules shows very high correspondence. For 125 out of 129 residues (excluding only the chain termini, residues 1 to 2 and 128 to 129) the root-mean-square (r.m.s.) deviation in main-chain atom positions is 0.27 A. For other structural parameters r.m.s. deviations are also low; torsion angles 6.5 degrees, hydrogen bond lengths 0.12 A, bonds to copper 0.04 A and bond angles at the copper 3.9 degrees. The only significant differences are at the chain termini and in several loops. Some of these can be attributed to crystal packing effects, others to genuine structural microheterogeneity. Refinement has confirmed that the copper co-ordination is best described as distorted trigonal planar, with strong in-plane bonds to His46 N delta 1, His117 N delta 1 and Cys112 S gamma, and much weaker axial interactions with Met121 S delta and Gly45 C = O. Two N-H...S hydrogen bonds characterize Cys112 S gamma as a thiolate (S-) sulphur and may influence the visible absorption maximum. Atoms in and around the copper site have very low mobility, whereas the most mobile regions of the molecule are the chain termini and some of the connecting loops between secondary structure elements, especially those at the "southern" end, remote from the copper site. Main-chain to side-chain hydrogen bonds supply important stabilizing interactions at the "northern" end. Surface features include the hydrophobic patch around His117, probably important for electron transfer, the SO2-4 site at His83, and the general absence of ion pairs, despite the presence of many charged amino acid residues. The 281 water molecules include 182 that occur as approximately twofold-related pairs. There are no internal water molecules. The water sites common to both azurin molecules include those in surface pockets and some in intermolecular contact regions. They are characterized by relatively low thermal parameters and numerous protein contacts.     

Lysozyme revisited: crystallographic evidence for distortion of an N-acetylmuramic acid residue bound in site D.

A structure of the trisaccharide 2-acetamido-2-deoxy-D-muramic acid-beta (1----4)-2-acetamido-2-deoxy-D-glucose-beta (1----4)-2-acetamido-2-deoxy-D-muramic acid (NAM-NAG-NAM), bound to subsites B, C and D in the active-site cleft of hen egg-white lysozyme has been determined and refined at 1.5 A resolution. The resulting atomic co-ordinates indicate that the NAM residue in site D is distorted from the full 4C1 chair conformation to one in which the ring atoms C-1, C-2, O-5 and C-5 are approximately coplanar, and the hydroxymethyl group is positioned axially (a conformation best described as a sofa). This finding supports the original proposals that suggested the ground-state conformation of the sugar bound in site D is strained to one that more closely resembles the geometry required for the oxocarbonium-ion transition state, the next step along the reaction pathway. Additionally, detailed analysis at 1.5 A resolution of the environments of the catalytic residues Glu35 and Asp52 provides new information on the properties that may allow lysozyme to promote the stabilization of an unusually long-lived oxocarbonium-ion transition state. Intermolecular interactions between the N-acetylmuramic acid residue in site D and the lysozyme molecule that contribute to the saccharide ring distortion include: close packing of the O-3' lactyl group with a hydrogen-bonded "platform" of enzyme residues (Asp52, Asn46, Asn59, Ser50 and Asp48), a close contact between the hydroxymethyl group of ring D and the 2'-acetamido group of ring C and a strong hydrogen-bonded interaction between the NH group of Val109 and O-6 of ring D that stabilizes the observed quasi-axial orientation of the -CH2OH group. Additionally, the structure of this complex shows a strong hydrogen bond between the carboxyl group of Glu35 and the beta-anomeric hydroxyl group of the NAM residue in site D. The hydrogen-bonded environment of Asp52 in the native enzyme and in the complex coupled with the very unfavorable direction of approach of the potential carboxylate nucleophile makes it most unlikely that there is a covalent glycosylenzyme intermediate on the hydrolysis pathway of hen egg-white lysozyme.     

OPUS-PSP: an orientation-dependent statistical all-atom potential derived from side-chain packing

Here we report an orientation-dependent statistical all-atom potential derived from side-chain packing, named OPUS-PSP. It features a basis set of 19 rigid-body blocks extracted from the chemical structures of all 20 amino acid residues. The potential is generated from the orientation-specific packing statistics of pairs of those blocks in a non-redundant structural database. The purpose of such an approach is to capture the essential elements of orientation dependence in molecular packing interactions. Tests of OPUS-PSP on commonly used decoy sets demonstrate that it significantly outperforms most of the existing knowledge-based potentials in terms of both its ability to recognize native structures and consistency in achieving high Z-scores across decoy sets. As OPUS-PSP excludes interactions among main-chain atoms, its success highlights the crucial importance of side-chain packing in forming native protein structures. Moreover, OPUS-PSP does not explicitly include solvation terms, and thus the potential should perform well when the solvation effect is difficult to determine, such as in membrane proteins. Overall, OPUS-PSP is a generally applicable potential for protein structure modeling, especially for handling side-chain conformations, one of the most difficult steps in high-accuracy protein structure prediction and refinement.     

Relation between sequence similarity and structural similarity in proteins. Role of important properties of amino acids

In a previous paper we obtained ten (orthogonal) factors, linear combinations of which can express the properties of the 20 naturally occurring amino acids. In this paper, we assume that the most important properties (linear combinations of these ten factors) that determine the three-dimensional structure of a protein are conserved properties, i.e., are those that have been conserved during evolution. Two definitions of a conserved property are presented: (1) a conserved property for an average protein is defined as that linear combination of the ten factors that optimally expresses the similarity of one amino acid to another (hence, little change during evolution), as given by the relatedness odds matrix of Dayhoff et al.; (2) a conserved property for each position in the amino acid sequence (locus) of a specific family of homologous proteins (the cytochromec family or the globin family) is defined as that linear combination of the ten factors that is common among a set of amino acids at a given locus when the sequences are properly aligned. When the specificity at each locus is averaged over all loci, the same features are observed for three expressions of these two definitions, namely the conserved property for an average protein, the average conserved property for the cytochromec family, and the average conserved property for the globin family; we find that bulk and hydrophobicity (information about packing and long-range interactions) are more important than other properties, such as the preference for adopting a specific backbone structure (information about short-range interactions). We also demonstrate that the sequence profile of a conserved property, defined for each locus of a protein family (definition 2), corresponds uniquely to the three-dimensional structure, while the conserved property for an average protein (definition 1) is not useful for the prediction of protein structure. The amino acid sequences of numerous proteins are searched to find those that are similar, in terms of the conserved properties (definition 2), to sequences of the same size from one of the homologous families (cytochromec and globin, respectively) for whose loci the conserved properties were defined. Many similar sequences are found, the number of similarities decreasing with increasing size of the segment. However, the segments must be rather long (15 residues) before the comparisons become meaningful. As an example, one sufficiently large sequence (20 residues) from a protein of known structure (apo-liver alcohol dehydrogenase that is not a member of either family) is found to be similar in the conserved properties to a particular sequence of a member of the family of human hemoglobin chains, and the two sequences have similar structures. This means that, since conserved properties are expected to be structure determinants, we can use the conserved properties to predict an initial protein structure for subsequent energy minimization for a protein for which the conserved properties are similar to those of a family of proteins with a sufficiently large number of homologous amino acid sequences; such a large number of homologous sequences is required to define a conserved property for each locus of the homologous protein family.     

Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions.

The MAT alpha 2 homeodomain regulates the expression of cell type-specific genes in yeast. We have determined the 2.7 A resolution crystal structure of the alpha 2 homeodomain bound to a biologically relevant DNA sequence. The DNA in this complex is contacted primarily by the third of three alpha-helices, with additional contacts coming from an N-terminal arm. Comparison of the yeast alpha 2 and the Drosophila engrailed homeodomain-DNA complexes shows that the protein fold is highly conserved, despite a 3-residue insertion in alpha 2 and only 27% sequence identity between the two homeodomains. Moreover, the orientation of the recognition helix on the DNA is also conserved. This docking arrangement is maintained by side chain contacts with the DNA--primarily the sugar-phosphate backbone--that are identical in alpha 2 and engrailed. Since these residues are conserved among all homeodomains, we propose that the contacts with the DNA are also conserved and suggest a general model for homeodomain-DNA interactions.     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

DNA-induced partial unfolding of prion protein leads to its polymerisation to amyloid

The full-length mouse recombinant prion protein (23-231 amino acid residues) contains all of its structural elements viz. three alpha-helices and a short two-stranded antiparallel beta-sheet in its C-terminal fragment comprising 121-231 amino acid residues. The incubated mixture of this prion protein fragment and nucleic acid results in the formation of amyloid fibres evidenced from electron microscopy, birefringence and fluorescence of the fibre bound Congo Red and Thioflavin T dyes, respectively. The secondary structure of the amyloid formed in nucleic acid solution is similar to the in vivo isolated prion protein 27-30 amyloid but unlike in it, a hydrophobic milieu is absent in the 121-231 amyloid. Thermal denaturation study demonstrates a partial unfolding of the protein fragment in nucleic acid solution. We propose that nucleic acid catalyses unfolding of prion protein helix 1 followed by a nucleation-dependent polymerisation of the protein to amyloid.     

Topological analysis of the complex formed between neurokinin A and the NK2 tachykinin receptor

Neurokinin A stimulates physiological responses in the peripheral and central nervous systems upon interacting primarily with the tachykinin NK2 receptor (NK2R). In this study, the structure of NKA bound to the NK2R is characterised by use of fluorescence resonance energy transfer. Four fluorescent NKA analogues with Texas red introduced at amino acid positions 1, 4, 7 and 10 were prepared. When bound to a NK2R carrying enhanced green fluorescent protein at the N-terminus, all peptides reduce green fluorescent protein fluorescence from 10% to 50% due to energy transfer. The derived donor-acceptor distances are 46, 55, 59 and 69 A for the fluorophore linked to positions 1-10, respectively. The monotonic increase in distance clearly indicates that the peptide adopts an extended structure when bound to its receptor. The present data are used, in combination with rhodopsin structure, fluorescence studies, photoaffinity labelling and site-directed mutagenesis data to design a computer model of the NKA-NK2R complex. We propose that the N-terminus of NKA is exposed and accessible to the extracellular medium. Subsequent amino acids of the NKA peptide become progressively more buried residues up to approximately one-third of the transmembrane-spanning domain.     

Achieving stability and conformational specificity in designed proteins via binary patterning

We have developed a method to determine the optimal binary pattern (arrangement of hydrophobic and polar amino acids) of a target protein fold prior to amino acid sequence selection in protein design studies. A solvent accessible surface is generated for a target fold using its backbone coordinates and "generic" side-chains, which are constructs whose size and shape are similar to an average amino acid. Each position is classified as hydrophobic or polar according to the solvent exposure of its generic side-chain. The method was tested by analyzing a set of proteins in the Protein Data Bank and by experimentally constructing and analyzing a set of engrailed homeodomain variants whose binary patterns were systematically varied. Selection of the optimal binary pattern results in a designed protein that is monomeric, well-folded, and hyperthermophilic. Homeodomain variants with fewer hydrophobic residues are destabilized, while additional hydrophobic residues induce aggregation. Binary patterning, in conjunction with a force field that models folded state energies, appears sufficient to satisfy two basic goals of protein design: stability and conformational specificity.     

Crystal structures of CheY from Thermotoga maritima do not support conventional explanations for the structural basis of enhanced thermostability.

The crystal structure of CheY protein from Thermotoga maritima has been determined in four crystal forms with and without Mg++ bound, at up to 1.9 A resolution. Structural comparisons with CheY from Escherichia coli shows substantial similarity in their folds, with some concerted changes propagating away from the active site that suggest how phosphorylated CheY, a signal transduction protein in bacterial chemotaxis, is recognized by its targets. A highly conserved segment of the protein (the "y-turn loop," residues 55-61), previously suggested to be a rigid recognition determinant, is for the first time seen in two alternative conformations in the different crystal structures. Although CheY from Thermotoga has much higher thermal stability than its mesophilic counterparts, comparison of structural features previously proposed to enhance thermostability such as hydrogen bonds, ion pairs, compactness, and hydrophobic surface burial would not suggest it to be so.     

The structure and function of omega loop A replacements in cytochrome c.

The structural and functional consequences of replacing omega-loop A (residues 18-32) in yeast iso-1-cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined. The three-dimensional structure of this loop replacement mutant RepA2 cytochrome c, and a second mutant RepA2(Val 20) cytochrome c in which residue 20 was back substituted to valine, were determined using X-ray diffraction techniques. A change in the molecular packing is evident in the RepA2 mutant protein, which has a phenylalanine at position 20, a residue considerably larger than the valine found in wild-type yeast iso-1-cytochrome c. The side chain of Phe 20 is redirected toward the molecular surface, altering the packing of this region of omega-loop A with the hydrophobic core of the protein. In the RepA2(Val 20) structure, omega-loop A contains a valine at position 20, which restores the original wild-type packing arrangement of the hydrophobic core. Also, as a result of omega-loop A replacement, residue 26 is changed from a histidine to asparagine, which results in displacements of the main-chain atoms near residue 44 to which residue 26 is hydrogen bonded. In vivo studies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2(Val 20) cytochrome c behaves much like the wild-type yeast iso-1 protein, whereas the stability and function of the RepA2 cytochrome c showed a temperature dependence. The midpoint reduction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25 degrees C. This is 19 mV less than the wild-type and RepA2(Val 20) proteins (290 mV) and may result from disruption of the hydrophobic packing in the heme pocket and increased mobility of omega-loop A in RepA2 cytochrome c. The temperature dependence of the reduction potential is also greatly enhanced in the RepA2 protein.     

Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineering

Outer surface protein A (OspA) from Borrelia burgdorferi has an unusual dumbbell-shaped structure in which two globular domains are connected with a "single-layer" beta-sheet (SLB). The protein is highly soluble, and it has been recalcitrant to crystallization. Only OspA complexes with Fab fragments have been successfully crystallized. OspA contains a large number of Lys and Glu residues, and these "high entropy" residues may disfavor crystal packing because some of them would need to be immobilized in forming a crystal lattice. We rationally designed a total of 13 surface mutations in which Lys and Glu residues were replaced with Ala or Ser. We successfully crystallized the mutant OspA without a bound Fab fragment and extended structure analysis to a 1.15 Angstroms resolution. The new high-resolution structure revealed a unique backbone hydration pattern of the SLB segment in which water molecules fill the "weak spots" on both faces of the antiparallel beta-sheet. These well-defined water molecules provide additional structural links between adjacent beta-strands, and thus they may be important for maintaining the rigidity of the SLB that inherently lacks tight packing afforded by a hydrophobic core. The structure also revealed new information on the side-chain dynamics and on a solvent-accessible cavity in the core of the C-terminal globular domain. This work demonstrates the utility of extensive surface mutation in crystallizing recalcitrant proteins and dramatically improving the resolution of crystal structures, and provides new insights into the stabilization mechanism of OspA.