Experimental evidence for the correlation of bond distances in peptide groups detected in ultrahigh-resolution protein structures

The structural analysis of a deamidated derivative of ribonuclease A, determined at 0.87 A resolution, reveals a highly significant negative correlation between CN and CO bond distances in peptide groups. This trend, i.e. the CO bond lengthens when the CN bond shortens, is also found in seven out of eight protein structures, determined at ultrahigh resolution (<0.95 A). In five of them the linear correlation is statistically significant at the 95% confidence level. The present findings are consistent with the traditional view of amide resonance and, although already found in small peptide structures, they represent a new and important result. In fact, in a protein structure the fine details of the peptide geometry are only marginally affected by the crystal field and they are mostly produced by intramolecular and solvent interactions. The analysis of very high-resolution protein structures can reveal subtle information about local electronic features of proteins which may be critical to folding, function or ligand binding.     

Contact order and ab initio protein structure prediction

Although much of the motivation for experimental studies of protein folding is to obtain insights for improving protein structure prediction, there has been relatively little connection between experimental protein folding studies and computational structural prediction work in recent years. In the present study, we show that the relationship between protein folding rates and the contact order (CO) of the native structure has implications for ab initio protein structure prediction. Rosetta ab initio folding simulations produce a dearth of high CO structures and an excess of low CO structures, as expected if the computer simulations mimic to some extent the actual folding process. Consistent with this, the majority of failures in ab initio prediction in the CASP4 (critical assessment of structure prediction) experiment involved high CO structures likely to fold much more slowly than the lower CO structures for which reasonable predictions were made. This bias against high CO structures can be partially alleviated by performing large numbers of additional simulations, selecting out the higher CO structures, and eliminating the very low CO structures; this leads to a modest improvement in prediction quality. More significant improvements in predictions for proteins with complex topologies may be possible following significant increases in high-performance computing power, which will be required for thoroughly sampling high CO conformations (high CO proteins can take six orders of magnitude longer to fold than low CO proteins). Importantly for such a strategy, simulations performed for high CO structures converge much less strongly than those for low CO structures, and hence, lack of simulation convergence can indicate the need for improved sampling of high CO conformations. The parallels between Rosetta simulations and folding in vivo may extend to misfolding: The very low CO structures that accumulate in Rosetta simulations consist primarily of local up-down beta-sheets that may resemble precursors to amyloid formation.     

Production and characterization of recombinant perdeuterated cholesterol oxidase

Cholesterol oxidase (CO) is a FAD (flavin adenine dinucleotide) containing enzyme that catalyzes the oxidization and isomerization of cholesterol. Studies directed toward elucidating the catalytic mechanism of CO will provide an important general understanding of Flavin-assisted redox catalysis. Hydrogen atoms play an important role in enzyme catalysis; however, they are not readily visualized in protein X-ray diffraction structures. Neutron crystallography is an ideal method for directly visualizing hydrogen positions at moderate resolutions because hydrogen and deuterium have comparable neutron scattering lengths to other heavy atoms present in proteins. The negative coherent and large incoherent scattering lengths of hydrogen atoms in neutron diffraction experiments can be circumvented by replacing hydrogen atoms with its isotope, deuterium. The perdeuterated form of CO was successfully expressed from minimal medium, purified, and crystallized. X-ray crystallographic structures of the enzyme in the perdeuterated and hydrogenated states confirm that there are no apparent structural differences between the two enzyme forms. Kinetic assays demonstrate that perdeuterated and hydrogenated enzymes are functionally identical. Together, structural and functional studies indicate that the perdeuterated protein is suitable for structural studies by neutron crystallography directed at understanding the role of hydrogen atoms in enzyme catalysis.     

The relation of ion pairs to protein hydration: An IR spectroscopic and X-ray crystallographic survey

We report here a novel ir technique that we use to determine the apparent van't Hoff's energy for the distillation of water from protein amide and acid groups. The method is used to study the hydration properties of ten different proteins. The results show that water removal from the environment of ion-paired acid groups (those ? 4 Å from a basic group on the protein surface) requires less energy than water removal from the environment of nonpaired acid groups. These observations are supported by an analysis of the solvent distributions in protein crystal structures. A model is proposed for the interaction between water and protein acid groups, and this model is used to account for the distributions and properties of certain eye-lens proteins.     

Contact potential that recognizes the correct folding of globular proteins.

We have devised a continuous function of interresidue contacts in globular proteins such that the X-ray crystal structure has a lower function value than that of thousands of protein-like alternative conformations. Although we fit the adjustable parameters of the potential using only 10,000 alternative structures for a selected training set of 37 proteins, a grand total of 530,000 constraints was satisfied, derived from 73 proteins and their numerous alternative conformations. In every case where the native conformation is adequately globular and compact, according to objective criteria we have developed, the potential function always favors the native over all alternatives by a substantial margin. This is true even for an additional three proteins never used in any way in the fitting procedure. Conformations differing only slightly from the native, such as those coming from crystal structures of the same protein complexed with different ligands or from crystal structures of point mutants, have function values very similar to the native's and always less than those of alternatives derived from substantially different crystal structures. This holds for all 95 structures that are homologous to one or another of various proteins we used. Realizing that this potential should be useful for modeling the conformation of new protein sequences from the body of protein crystal structures, we suggest a test for deciding whether a nearly correct approximation to the native conformation has been found.     

Statistical analysis of interface similarity in crystals of homologous proteins

Many proteins function as homo-oligomers and are regulated via their oligomeric state. For some proteins, the stoichiometry of homo-oligomeric states under various conditions has been studied using gel filtration or analytical ultracentrifugation experiments. The interfaces involved in these assemblies may be identified using cross-linking and mass spectrometry, solution-state NMR, and other experiments. However, for most proteins, the actual interfaces that are involved in oligomerization are inferred from X-ray crystallographic structures using assumptions about interface surface areas and physical properties. Examination of interfaces across different Protein Data Bank (PDB) entries in a protein family reveals several important features. First, similarities in space group, asymmetric unit size, and cell dimensions and angles (within 1%) do not guarantee that two crystals are actually the same crystal form, containing similar relative orientations and interactions within the crystal. Conversely, two crystals in different space groups may be quite similar in terms of all the interfaces within each crystal. Second, NMR structures and an existing benchmark of PDB crystallographic entries consisting of 126 dimers as well as larger structures and 132 monomers were used to determine whether the existence or lack of common interfaces across multiple crystal forms can be used to predict whether a protein is an oligomer or not. Monomeric proteins tend to have common interfaces across only a minority of crystal forms, whereas higher-order structures exhibit common interfaces across a majority of available crystal forms. The data can be used to estimate the probability that an interface is biological if two or more crystal forms are available. Finally, the Protein Interfaces, Surfaces, and Assemblies (PISA) database available from the European Bioinformatics Institute is more consistent in identifying interfaces observed in many crystal forms compared with the PDB and the European Bioinformatics Institute's Protein Quaternary Server (PQS). The PDB, in particular, is missing highly likely biological interfaces in its biological unit files for about 10% of PDB entries.     

NMR assignments, secondary structure, and global fold of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea

Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-hand calcium-binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum.     

Crystal structure of a protein associated with cell division from Mycoplasma pneumoniae (GI: 13508053): a novel fold with a conserved sequence motif

UPF0040 is a family of proteins implicated in a cellular function of bacteria cell division. There is no structure information available on protein of this family. We have determined the crystal structure of a protein from Mycoplasma pneumoniae that belongs to this family using X-ray crystallography. Structural homology search reveals that this protein has a novel fold with no significant similarity to any proteins of known three-dimensional structure. The crystal structures of the protein in three different crystal forms reveal that the protein exists as a ring of octamer. The conserved protein residues, including a highly conserved DXXXR motif, are examined on the basis of crystal structure.     

Analysis of solvent content and oligomeric states in protein crystals—does symmetry matter?

A nonredundant set of 9081 protein crystal structures in the Protein Data Bank was used to examine the solvent content, the number of polypeptide chains, and the oligomeric states of proteins in crystals as a function of crystal symmetry (as classified by crystal systems and space groups). It was found that there is a correlation between solvent content and crystal symmetry. Surprisingly, proteins crystallizing in lower symmetry systems have lower solvent content compared to those crystallizing in higher symmetry systems. Nevertheless, there is no universal correlation between solvent content and preferences of macromolecules to crystallize in certain space groups. Crystal symmetry as a function of oligomeric state was examined, where trimers, tetramers, and hexamers were found to prefer to crystallize in systems where the oligomer symmetry could be incorporated in the crystal symmetry. Our analysis also shows that the frequency distribution within the enantiomorphous pairs of space groups does not differ significantly, in contrast to previous reports.     

Structure-Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility

Advances reported over the last few years and the increasing availability of protein crystal structure data have greatly improved structure-based druggability approaches. However, in practice, nearly all druggability estimation methods are applied to protein crystal structures as rigid proteins, with protein flexibility often not directly addressed. The inclusion of protein flexibility is important in correctly identifying the druggability of pockets that would be missed by methods based solely on the rigid crystal structure. These include cryptic pockets and flexible pockets often found at protein-protein interaction interfaces. Here, we apply an approach that uses protein modeling in concert with druggability estimation to account for light protein backbone movement and protein side-chain flexibility in protein binding sites. We assess the advantages and limitations of this approach on widely-used protein druggability sets. Applying the approach to all mammalian protein crystal structures in the PDB results in identification of 69 proteins with potential druggable cryptic pockets.     

Fluctuations in ion pairs and their stabilities in proteins

This report investigates the effect of systemic protein conformational flexibility on the contribution of ion pairs to protein stability. Toward this goal, we use all NMR conformer ensembles in the Protein Data Bank (1) that contain at least 40 conformers, (2) whose functional form is monomeric, (3) that are nonredundant, and (4) that are large enough. We find 11 proteins adhering to these criteria. Within these proteins, we identify 22 ion pairs that are close enough to be classified as salt bridges. These are identified in the high-resolution crystal structures of the respective proteins or in the minimized average structures (if the crystal structures are unavailable) or, if both are unavailable, in the "most representative" conformer of each of the ensembles. We next calculate the electrostatic contribution of each such ion pair in each of the conformers in the ensembles. This results in a comprehensive study of 1,201 ion pairs, which allows us to look for consistent trends in their electrostatic contributions to protein stability in large sets of conformers. We find that the contributions of ion pairs vary considerably among the conformers of each protein. The vast majority of the ion pairs interconvert between being stabilizing and destabilizing to the structure at least once in the ensembles. These fluctuations reflect the variabilities in the location of the ion pairing residues and in the geometric orientation of these residues, both with respect to each other, and with respect to other charged groups in the remainder of the protein. The higher crystallographic B-factors for the respective side-chains are consistent with these fluctuations. The major conclusion from this study is that salt bridges observed in crystal structure may break, and new salt bridges may be formed. Hence, the overall stabilizing (or, destabilizing) contribution of an ion pair is conformer population dependent.     

Crystal structures of deoxy and CO-bound bjFixLH reveal details of ligand recognition and signaling

Rhizobia directly regulate the expression of genes required for symbiotic nitrogen fixation in response to oxygen concentration via the sensor protein FixL. The N-terminal PAS domain of FixL contains a histidine-coordinated heme and regulates the activity of its effector domain, a C-terminal histidine kinase, in response to binding of oxygen and other ligands at the heme. To further investigate ligand-induced inhibition of FixL, we have determined the crystal structures of the heme domain in both the deoxy state and bound to carbon monoxide, a weak inhibitor of FixL kinase activity. Structures collected at room temperature are presented in each state from two crystallographic space groups at 1.8 and 2 A resolution. These structures reveal displacement of the residues of the H(beta) and I(beta) strands by Leu236 upon CO binding, and this structural change propagates more than 15 A to a region of the structure implicated in signal transduction in PAS proteins. Displacement of residues Ile215, Ile216, and Gly217 in the FG loop is also evident, accompanied by the movement of heme propionate 6 upon change in iron ligation. CO binding increases the temperature factors in the FG loop of the protein and disorders the side chain of Arg206, a conserved residue involved in the FG loop switch mechanism. We relate these results to structural changes in other PAS sensor domains and their involvement in catalytic control.     

Myoglobin models and steric origins of the discrimination between O2 and CO

 Synthetic models of the myoglobin active site have provided much insight into factors that affect CO and O₂ binding in the proteins. "Capped" and "pocket" metal porphyrin systems have been developed to probe how steric factors affect ligand binding and ultimately to elucidate important aspects of the mechanism of CO discrimination in the proteins. These model porphyrins are among the most thoroughly characterized systems to date. From the twenty-one known crystal structures, analysis of the types of distortion that occur upon ligand binding under the cap, including porphyrin doming and ruffling, lateral and horizontal movement of the cap, and bending and tilting of the Fe–C–O bond, provides an indication of how steric interactions will affect structure in Hb and Mb. The model porphyrin systems discussed range from those that discriminate against O₂ binding compared to biological systems to those with similar CO and O₂ binding strength to myoglobin, and also to those that bind both O₂ and CO very weakly or not at all. The primary type of distortion observed upon CO binding is vertical or lateral movement of the cap and some ruffling of the porphyrin plane. Minimal bending or tilting of the M–C–O bond is observed, suggesting that the Fe–C–O bending that has been found from crystal structures of the hemoproteins is unlikely. Received, accepted: 23 May 1997     

Interaction geometry involving planar groups in protein-protein interfaces

The geometry of interactions of planar residues is nonrandom in protein tertiary structures and gives rise to conventional, as well as nonconventional (X--H...pi, X--H...O, where X = C, N, or O) hydrogen bonds. Whether a similar geometry is maintained when the interaction is across the protein-protein interface is addressed here. The relative geometries of interactions involving planar residues, and the percentage of contacts giving rise to different types of hydrogen bonds are quite similar in protein structures and the biological interfaces formed by protein chains in homodimers and protein-protein heterocomplexes--thus pointing to the similarity of chemical interactions that occurs during protein folding and binding. However, the percentage is considerably smaller in the nonspecific and nonphysiological interfaces that are formed in crystal lattices of monomeric proteins. The C--H...O interaction linking the aromatic and the peptide groups is quite common in protein structures as well as the three types of interfaces. However, as the interfaces formed by crystal contacts are depleted in aromatic residues, the weaker hydrogen bond interactions would contribute less toward their stability.     

Contact order revisited: influence of protein size on the folding rate

Guided by the recent success of empirical model predicting the folding rates of small two-state folding proteins from the relative contact order (CO) of their native structures, by a theoretical model of protein folding that predicts that logarithm of the folding rate decreases with the protein chain length L as L(2/3), and by the finding that the folding rates of multistate folding proteins strongly correlate with their sizes and have very bad correlation with CO, we reexamined the dependence of folding rate on CO and L in attempt to find a structural parameter that determines folding rates for the totality of proteins. We show that the Abs_CO = CO x L, is able to predict rather accurately folding rates for both two-state and multistate folding proteins, as well as short peptides, and that this Abs_CO scales with the protein chain length as L(0.70 +/- 0.07) for the totality of studied single-domain proteins and peptides.     

Crystal structures of MBP fusion proteins

Although chaperone‐assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose‐binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter‐domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP‐mediated structural distortions are very rare.     

Crystal structures of the signal transducing protein GlnK from Thermus thermophilus HB8

The Thermus thermophilus HB8 genome encodes a signal transducing PII protein, GlnK. The crystal structures of GlnK have been determined in two different space groups, P2(1)2(1)2(1) and P3(1)21. The PII protein has the T-loop, which is essential for interactions with receptor proteins. In both crystal forms, three GlnK molecules form a trimer in the asymmetric unit. In one P2(1)2(1)2(1) crystal form, the three T-loops in the trimer are disordered, while in another P2(1)2(1)2(1) crystal form, the T-loop from one molecule in the trimer is ordered. In the P3(1)21 crystal, one T-loop is ordered while the other two T-loops are disordered. The conformations of the ordered T-loops significantly differ between the two crystal forms; one makes the alpha-helix in the middle of the T-loop, while the other has an extension of the beta-hairpin. Two different conformations are captured by the crystal contacts. The observation of multiple T-loop conformations suggests that the T-loop could potentially exhibit "polysterism," which would be important for interactions with receptor proteins. The crystal structures of the nucleotide-bound forms, GlnK.ATP and GlnK.ADP, have also been determined. ATP/ADP binding within a cleft at the interface of two adjacent T. thermophilus GlnK monomers might affect the conformation of the T-loop.     

Structures of designed armadillo repeat proteins binding to peptides fused to globular domains

Designed armadillo repeat proteins (dArmRP) are α‐helical solenoid repeat proteins with an extended peptide binding groove that were engineered to develop a generic modular technology for peptide recognition. In this context, the term “peptide” not only denotes a short unstructured chain of amino acids, but also an unstructured region of a protein, as they occur in termini, loops, or linkers between folded domains. Here we report two crystal structures of dArmRPs, in complex with peptides fused either to the N‐terminus of Green Fluorescent Protein or to the C‐terminus of a phage lambda protein D. These structures demonstrate that dArmRPs bind unfolded peptides in the intended conformation also when they constitute unstructured parts of folded proteins, which greatly expands possible applications of the dArmRP technology. Nonetheless, the structures do not fully reflect the binding behavior in solution, that is, some binding sites remain unoccupied in the crystal and even unexpected peptide residues appear to be bound. We show how these differences can be explained by restrictions of the crystal lattice or the composition of the crystallization solution. This illustrates that crystal structures have to be interpreted with caution when protein–peptide interactions are characterized, and should always be correlated with measurements in solution.