Structure of the YibK methyltransferase from Haemophilus influenzae (HI0766): a cofactor bound at a site formed by a knot

Despite the suitability of various lattice geometries for coarse-grained modeling of proteins, the actual packing geometry of residues in folded structures has remained largely unexplored. A strong tendency to assume a regular packing geometry is shown here by optimally reorienting and superimposing clusters of neighboring residues from databank structures examined on a coarse-grained (single-site-per-residue) scale. The orientation function (or order parameter) of the examined coordination clusters with respect to fcc lattice directions is found to be 0.82. The observed geometry, which may be termed an incomplete distorted face-centered cubic (fcc) packing, is apparently favored by the drive to maximize packing density, in a fashion analogous to the way identical spheres pack densely and follow fcc geometry. About 2/3 of all residues obey this packing geometry, while the remainder occupy other context-dependent positions. The preferred coordination directions show relatively small variations over the various amino acid types, consistent with uniform residue viewpoint. Both the extremes of solvent-exposed and completely buried residue neighborhoods approximate the same generic packing, the only difference being in the numbers (and not the orientations) of coordination sites that are occupied (or left void for solvent occupancy). We observe the prevalence of a rather uniform (tight) residue packing density throughout the structure, including even the residues packed near solvent-exposed regions. The observed orientation distribution reveals an underlying, intrinsic orientation lattice for proteins.     

Influence of the C-terminus of the glycophorin A transmembrane fragment on the dimerization process

The monomer-dimer equilibrium of the glycophorin A (GpA) transmembrane (TM) fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix-helix packing in a membrane-mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C-terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement of TM fragments that takes place when leucine repeats are introduced in such protein segments. We also show that secondary structure of GpA derivatives was independent from dimerization, in agreement with the two-stage model for membrane protein folding and oligomerization.     

Understanding the recognition of protein structural classes by amino acid composition

Knowledge of amino acid composition, alone, is verified here to be sufficient for recognizing the structural class, α, β, α+β, or α/β of a given protein with an accuracy of 81%. This is supported by results from exhaustive enumerations of all conformations for all sequences of simple, compact lattice models consisting of two types (hydrophobic and polar) of residues. Different compositions exhibit strong affinities for certain folds. Within the limits of validity of the lattice models, two factors appear to determine the choice of particular folds: 1) the coordination numbers of individual sites and 2) the size and geometry of non-bonded clusters. These two properties, collectively termed the distribution of non-bonded contacts, are quantitatively assessed by an eigenvalue analysis of the so-called Kirchhoff or adjacency matrices obtained by considering the non-bonded interactions on a lattice. The analysis permits the identification of conformations that possess the same distribution of non-bonded contacts. Furthermore, some distributions of non-bonded contacts are favored entropically, due to their high degeneracies. Thus, a competition between enthalpic and entropic effects is effective in determining the choice of a distribution for a given composition. Based on these findings, an analysis of non-bonded contacts in protein structures was made. The analysis shows that proteins belonging to the four distinct folding classes exhibit significant differences in their distributions of non-bonded contacts, which more directly explains the success in predicting structural class from amino acid composition. Proteins 29:172–185, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Goverment work and, as such, is in the public domain in the United States of America.

     

Stabilization of Bacillus subtilis Lipase A by increasing the residual packing

Introduction of well-packed residues to the interior of a protein structure could be considered as a stabilization strategy since the reduction of buried cavities might stabilize protein structure. In this study, the less-packed residues with no water-contact were selected as target sites for increasing residual packing. When Lipase A from Bacillus subtilis (179 amino acids) was used as a model system, 43 less-packed residues were initially considered by analyzing their residual packing value and residual exposure ratio. Among the 43 residues, small amino acids such as GLY and ALA were chosen as target sites. Packing increases of ALA to VAL and GLY to ALA were estimated, by molecular modeling, to give 0.5368∼0.7433 kcal mol^-1 stabilization. Mutants of Lipase A such as A38V, A75V, G80A, A105V A146V, and G172A were obtained via protein engineering. Thermostability assays revealed that A38V, G80A and G172V were the most stable mutants. This procedure for selecting the target residues for improved thermostability of Lipase A could be applied for improving the thermostability of other proteins and enzymes.     

Stabilization of Bacillus subtilis Lipase A by increasing the residual packing

Introduction of well-packed residues to the interior of a protein structure could be considered as a stabilization strategy since the reduction of buried cavities might stabilize protein structure. In this study, the less-packed residues with no water-contact were selected as target sites for increasing residual packing. When Lipase A from Bacillus subtilis (179 amino acids) was used as a model system, 43 less-packed residues were initially considered by analyzing their residual packing value and residual exposure ratio. Among the 43 residues, small amino acids such as GLY and ALA were chosen as target sites. Packing increases of ALA to VAL and GLY to ALA were estimated, by molecular modeling, to give 0.5368～0.7433?kcal mol^?1 stabilization. Mutants of Lipase A such as A38V, A75V, G80A, A105V A146V, and G172A were obtained via protein engineering. Thermostability assays revealed that A38V, G80A and G172V were the most stable mutants. This procedure for selecting the target residues for improved thermostability of Lipase A could be applied for improving the thermostability of other proteins and enzymes.     

An amino acid code for β‐sheet packing structure

To understand the relationship between protein sequence and structure, this work extends the knob‐socket model in an investigation of β‐sheet packing. Over a comprehensive set of β‐sheet folds, the contacts between residues were used to identify packing cliques: sets of residues that all contact each other. These packing cliques were then classified based on size and contact order. From this analysis, the two types of four‐residue packing cliques necessary to describe β‐sheet packing were characterized. Both occur between two adjacent hydrogen bonded β‐strands. First, defining the secondary structure packing within β‐sheets, the combined socket or XY:HG pocket consists of four residues i, i+2 on one strand and j, j+2 on the other. Second, characterizing the tertiary packing between β‐sheets, the knob‐socket XY:H+B consists of a three‐residue XY:H socket (i, i+2 on one strand and j on the other) packed against a knob B residue (residue k distant in sequence). Depending on the packing depth of the knob B residue, two types of knob‐sockets are found: side‐chain and main‐chain sockets. The amino acid composition of the pockets and knob‐sockets reveal the sequence specificity of β‐sheet packing. For β‐sheet formation, the XY:HG pocket clearly shows sequence specificity of amino acids. For tertiary packing, the XY:H+B side‐chain and main‐chain sockets exhibit distinct amino acid preferences at each position. These relationships define an amino acid code for β‐sheet structure and provide an intuitive topological mapping of β‐sheet packing. Proteins 2014; 82:2128–2140. © 2014 Wiley Periodicals, Inc.     

Calculation of protein volumes: An alternative to the Voronoi procedure

All the methods that have been applied to assessing local volume occupation or packing in proteins have particular defects. For example, the Voronoi method used by Richards (method A) and by Finney misallocates both non-bonded and covalent contacts in a geometrically rigorous, though chemically inconsistent, manner. Richards' method B, in which covalent and non-bonded contacts are partitioned in chemically sensible ways, is unfortunately not completely rigorous, in that every polyhedron vertex has associated with it a small vertex-error polyhedron, which is not allocated to any atom.We present here a generalization of the Voronoi method that is particularly suited to multicomponent assemblies such as proteins. This radical plane partitioning of volume is completely rigorous; it gives rise to no vertex error, and handles the more numerous non-bonded contacts realistically. Its application to RNAase-S is described and the results compared with both Voronoi's method and Richards' method B. A particular advantage of both the radical plane and Richards' methods is a relative insensitivity to the treatment of the surface, a problem that has plagued other approaches to describing packing in proteins. Although the radical plane is seen to misallocate volume chemically between covalently-bonded neighbours, this problem vanishes when groups of atoms in side-chain residues are considered.     

Calculation of standard atomic volumes for RNA and comparison with proteins: RNA is packed more tightly

Traditionally, for biomolecular packing calculations research has focused on proteins. Besides proteins, RNA is the other large biomolecule that has tertiary structure interactions and complex packing. No one has yet quantitatively investigated RNA packing or compared its packing to that of proteins because, until recently, there were no large RNA structures. Here we address this question in detail, using Voronoi volume calculations on a set of high-resolution RNA crystal structures. We do a careful parameterization, taking into account many factors such as atomic radii, crystal packing, structural complexity, solvent, and associated protein to obtain a self-consistent, universal set of volumes that can be applied to both RNA and protein. We report this set of volumes, which we call the NucProt parameter set. Our measured values are consistent across the many different RNA structures and packing environments. When common atom types are compared between proteins and RNA, nine of 12 types show that RNA has a smaller volume and packs more tightly than protein, suggesting that close-packing may be as important for the folding of RNAs as it is for proteins. Moreover, calculated partial specific volumes show that RNA bases pack more densely than corresponding aromatic residues from proteins. Finally, we find that RNA bases have similar packing volumes to DNA bases, despite the absence of tertiary contacts in DNA. Programs, parameter sets and raw data are available online at.     

Analysis of interactive packing of secondary structural elements in alpha/beta units in proteins

An alpha-helix and a beta-strand are said to be interactively packed if at least one residue in each of the secondary structural elements loses 10% of its solvent accessible contact area on association with the other secondary structural element. An analysis of all such 5,975 nonidentical alpha/beta units in protein structures, defined at < or = 2.5 A resolution, shows that the interaxial distance between the alpha-helix and the beta-strand is linearly correlated with the residue-dependent function, log[(V/nda)/n-int], where V is the volume of amino acid residues in the packing interface, nda is the normalized difference in solvent accessible contact area of the residues in packed and unpacked secondary structural elements, and n-int is the number of residues in the packing interface. The beta-sheet unit (beta u), defined as a pair of adjacent parallel or antiparallel hydrogen-bonded beta-strands, packing with an alpha-helix shows a better correlation between the interaxial distance and log(V/nda) for the residues in the packing interface. This packing relationship is shown to be useful in the prediction of interaxial distances in alpha/beta units using the interacting residue information of equivalent alpha/beta units of homologous proteins. It is, therefore, of value in comparative modeling of protein structures.     

Interaction of transmembrane helices by a knobs-into-holes packing characteristic of soluble coiled coils

Membrane-embedded protein domains frequently exist as α-helical bundles, as exemplified by photosynthetic reaction centers, bacteriorhodopsin, and cytochrome C oxidase. The sidechain packing between their transmembrane helices was investigated by a nearest-neighbor analysis which identified sets of interfacial residues for each analyzed helix–helix interface. For the left-handed helix–helix pairs, the interfacial residues almost exclusively occupy positions a, d, e, or g within a heptad motif (abcdefg) which is repeated two to three times for each interacting helical surface. The connectivity between the interfacial residues of adjacent helices conforms to the knobs-into-holes type of sidechain packing known from soluble coiled coils. These results demonstrate on a quantitative basis that the geometry of sidechain packing is similar for left-handed helix–helix pairs embedded in membranes and coiled coils of soluble proteins. The transmembrane helix–helix interfaces studied are somewhat less compact and regular as compared to soluble coiled coils and tolerate all hydrophobic amino acid types to similar degrees. The results are discussed with respect to previous experimental findings which demonstrate that specific interactions between transmembrane helices are important for membrane protein folding and/or oligomerization. Proteins 31:150–159, 1998. © 1998 Wiley-Liss, Inc.     

Modification of protein crystal packing by systematic mutations of surface residues: Implications on biotemplating and crystal porosity

Bioinspired nano‐scale biotemplating for the development of novel composite materials has recently culminated in several demonstrations of nano‐structured hybrid materials. Protein crystals, routinely prepared for the elucidation of protein 3D structures by X‐ray crystallography, present an ordered and highly accurate 3D array of protein molecules. Inherent to the 3D arrangement of the protein “building blocks” in the crystal, a complementary 3D array of interconnected cavities—voids array, exhibiting highly ordered porosity is formed. The porous arrays of protein crystal may serve as a nano‐structured, accurate biotemplate by a “filling” process. These cavities arrays are shaped by the mode of protein packing throughout the crystallization process. Here we propose and demonstrate feasibility of targeting site specific mutations to modify protein's surface to affect protein crystal packing, enabling the generation of a series of protein crystal “biotemplates” all originating from same parent protein. The selection of these modification sites was based on in silico analysis of protein–protein interface contact areas in the parent crystal. The model protein selected for this study was the N‐terminal type II cohesin from the cellulosomal scaffold in ScaB subunit of Acetivibrio cellulolyticus and mutations were focused on lysine residues involved in protein packing as prime target. The impact of systematically mutating these lysine residues on protein packing and its resulting interconnected cavities array were found to be most significant when surface lysine residues were substituted to tryptophan residues. Our results demonstrate the feasibility of using pre‐designed site directed mutations for the generation of a series of protein crystal biotemplates from a “parent” protein. Biotechnol. Bioeng. 2009; 104: 444–457 © 2009 Wiley Periodicals, Inc.     

An accurate feature-based method for identifying DNA-binding residues on protein surfaces

Proteins that interact with DNA play vital roles in all mechanisms of gene expression and regulation. In order to understand these activities, it is crucial to analyze and identify DNA-binding residues on DNA-binding protein surfaces. Here, we proposed two novel features B-factor and packing density in combination with several conventional features to characterize the DNA-binding residues in a well-constructed representative dataset of 119 protein-DNA complexes from the Protein Data Bank (PDB). Based on the selected features, a prediction model for DNA-binding residues was constructed using support vector machine (SVM). The predictor was evaluated using a 5-fold cross validation on above dataset of 123 DNA-binding proteins. Moreover, two independent datasets of 83 DNA-bound protein structures and their corresponding DNA-free forms were compiled. The B-factor and packing density features were statistically analyzed on these 83 pairs of holo-apo proteins structures. Finally, we developed the SVM model to accurately predict DNA-binding residues on protein surface, given the DNA-free structure of a protein. Results showed here indicate that our method represents a significant improvement of previously existing approaches such as DISPLAR. The observation suggests that our method will be useful in studying protein-DNA interactions to guide consequent works such as site-directed mutagenesis and protein-DNA docking.     

An amino acid code to define a protein's tertiary packing surface

One difficult aspect of the protein‐folding problem is characterizing the nonspecific interactions that define packing in protein tertiary structure. To better understand tertiary structure, this work extends the knob‐socket model by classifying the interactions of a single knob residue packed into a set of contiguous sockets, or a pocket made up of 4 or more residues. The knob‐socket construct allows for a symbolic two‐dimensional mapping of pockets. The two‐dimensional mapping of pockets provides a simple method to investigate the variety of pocket shapes to understand the geometry of protein tertiary surfaces. The diversity of pocket geometries can be organized into groups of pockets that share a common core, which suggests that some interactions in pockets are ancillary to packing. Further analysis of pocket geometries displays a preferred configuration that is right‐handed in α‐helices and left‐handed in β‐sheets. The amino acid composition of pockets illustrates the importance of nonpolar amino acids in packing as well as position specificity. As expected, all pocket shapes prefer to pack with hydrophobic knobs; however, knobs are not selective for the pockets they pack. Investigating side‐chain rotamer preferences for certain pocket shapes uncovers no strong correlations. These findings allow a simple vocabulary based on knobs and sockets to describe protein tertiary packing that supports improved analysis, design, and prediction of protein structure. Proteins 2016; 84:201–216. © 2015 Wiley Periodicals, Inc.     

Structural principles of alpha/beta barrel proteins: the packing of the interior of the sheet

Alpha/beta barrel structures very similar to that first observed in triose phosphate isomerase are now known to occur in 14 enzymes. To understand the origin of this fold, we analyzed in three of these proteins the geometry of the eight-stranded beta-sheets and the packing of the residues at the center of the barrel. The packing in this region is seen in its simplest form in glycolate oxidase. It consists of 12 residues arranged in three layers. Each layer contains four side chains. The packing of RubisCO and TIM can be understood in terms of distortions of this simple pattern, caused by residues with small side chains at some of the positions inside the barrel. Two classes of packing are found. In one class, to which RubisCO and TIM belong, the central layer is formed by a residue from the first, third, fifth, and seventh strands; the upper and lower layers are formed by residues from the second, fourth, sixth, and eighth strands. In the second class, to which GAO belongs, this is reversed: it is side chains from the even-numbered strands that form the central layer, and side chains from the odd-numbered strands that form the outer layers. Our results suggest that not all proteins with this fold are related by evolution, but that they represent a common favorable solution to the structural problems involved in the creation of a closed beta barrel.     

Occluded molecular surface: Analysis of protein packing

We describe a novel method to calculate the packing interactions in protein structural models. The method calculates the interatomic occluded surface areas for each atom in the protein model. The identification of, and degree of interaction with, neighboring atoms is accomplished by extending surface normal from a dot surface of each atom to the point of intersection with neighboring atoms. The combined occluded and non-occluded surface areas may be normalized for the amino acid composition of the protein providing a single parameter, the normalized protein surface ratio, which is diagnostic for native-like Structures. Individual residues in the model which are in infrequent occluded surface environments may be identified. The method provides a means to explicitly describe packing densities and packing environments of individual atoms in a protein model. Finally, the method allows estimation of the complementarity between any interacting molecules, for example a ligand binding to a receptor.     

De novo design of the hydrophobic core of ubiquitin.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.     

Energetics of the lattice: Packing elements in crystals of four-stranded intercalated cytosine-rich DNA molecules

Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice—these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine · cytosine⁺ (C · C⁺) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other—for example, when folded in more complicated higher order structures, such as mRNA and chromatin. © 1998 John Wiley & Sons, Inc. Biopoly 44: 257–267, 1997     

An amino acid packing code for α-helical structure and protein design

This work demonstrates that all packing in α-helices can be simplified to repetitive patterns of a single motif: the knob-socket. Using the precision of Voronoi Polyhedra/Delauney Tessellations to identify contacts, the knob-socket is a four-residue tetrahedral motif: a knob residue on one α-helix packs into the three-residue socket on another α-helix. The principle of the knob-socket model relates the packing between levels of protein structure: the intra-helical packing arrangements within secondary structure that permit inter-helix tertiary packing interactions. Within an α-helix, the three-residue sockets arrange residues into a uniform packing lattice. Inter-helix packing results from a definable pattern of interdigitated knob-socket motifs between two α-helices. Furthermore, the knob-socket model classifies three types of sockets: (1) free, favoring only intra-helical packing; (2) filled, favoring inter-helical interactions; and (3) non, disfavoring α-helical structure. The amino acid propensities in these three socket classes essentially represent an amino acid code for structure in α-helical packing. Using this code, we used a novel yet straightforward approach for the design of α-helical structure to validate the knob-socket model. Unique sequences for three peptides were created to produce a predicted amount of α-helical structure: mostly helical, some helical, and no helix. These three peptides were synthesized, and helical content was assessed using CD spectroscopy. The measured α-helicity of each peptide was consistent with the expected predictions. These results and analysis demonstrate that the knob-socket motif functions as the basic unit of packing and presents an intuitive tool to decipher the rules governing packing in protein structure.     

Variability of conformations at crystal contacts in BPTI represent true low-energy structures: correspondence among lattice packing and molecular dynamics structures.

The structures of five basic pancreatic trypsin inhibitor (BPTI) molecules are compared to establish the extent and nature of the conformational variability resulting from crystal packing effects. BPTI is an ideal system to evaluate such factors because of the availability of high resolution X-ray models of five different BPTI structures, each in a different crystal packing environment. Differences observed among the structures are found to be distributed throughout the molecule, although the regions that display most variability are associated with the loop structures (residues 14-17 and 24-29). The regions of structure that show the largest rms deviations from the mean of the five packing motifs correlate well with the presence of intermolecular contacts in the crystal lattice. For most of the molecules there is also a correspondence between a larger number of intermolecular contacts and systematically higher B-factors, although it is not apparent whether this is induced by the crystal contact or results from the fact that the contacts are made predominantly through surface loops. The conformational differences seen among the X-ray models constitute more than local shifts at the lattice contact surfaces, and in fact involve in some cases the making and breaking of intramolecular H-bonds. The magnitudes of the differences among packing models are significantly larger than those usually associated with changes induced by mutagenesis; for instance; the structural differences at the site of mutation observed on removing an internal disulfide from the molecule are significantly less than those associated with lattice contact effects. The crystal packing conformations are compared to representative structures of BPTI generated during a 96-psec molecular dynamics (MD) simulation. This comparison shows a high level of correspondence between the protein flexibility indicated by the X-ray and MD analyses, and specifically between those regions that are most variable. This suggests that the regions that show most variability among the crystal packing models are not artifacts of crystallization, but rather represent true low-energy conformers that have been preferentially selected by crystallization factors.