The finite number of global motion patterns available to symmetric protein complexes

In PDB, more than half of the entries are structure complexes and of these complexes, most are symmetric, composed of identical subunits. Complex formation is the way through which larger structures and even molecular machines are assembled and built in nature. In this work, we apply group theory and carry out a comprehensive study of the global motion patterns of protein complexes of various symmetries. The work presents for the first time a comprehensive list of all the symmetric, aesthetically pleasing, global motion patterns available to complexes of cyclic, dihedral, tetrahedral, or octahedral symmetry. Our results clearly demonstrate that complexes with the same symmetry will have the same global motion patterns and thus may function in a similar way, and that there are only a finite number of global motion patterns available to symmetric complexes as the number of protein symmetry groups is effectively finite. The work complements our current understanding of the principle of complex formation that has been mostly structure‐based by providing novel dynamics‐based insights. Furthermore, as dynamics is closely tied to function, these motion patterns can provide global insights into the general functional mechanisms of protein complexes.     

Benchmarking a computational design method for the incorporation of metal ion‐binding sites at symmetric protein interfaces

The design of novel metal‐ion binding sites along symmetric axes in protein oligomers could provide new avenues for metalloenzyme design, construction of protein‐based nanomaterials and novel ion transport systems. Here, we describe a computational design method, symmetric protein recursive ion‐cofactor sampling (SyPRIS), for locating constellations of backbone positions within oligomeric protein structures that are capable of supporting desired symmetrically coordinated metal ion(s) chelated by sidechains (chelant model). Using SyPRIS on a curated benchmark set of protein structures with symmetric metal binding sites, we found high recovery of native metal coordinating rotamers: in 65 of the 67 (97.0%) cases, native rotamers featured in the best scoring model while in the remaining cases native rotamers were found within the top three scoring models. In a second test, chelant models were crossmatched against protein structures with identical cyclic symmetry. In addition to recovering all native placements, 10.4% (8939/86013) of the non‐native placements, had acceptable geometric compatibility scores. Discrimination between native and non‐native metal site placements was further enhanced upon constrained energy minimization using the Rosetta energy function. Upon sequence design of the surrounding first‐shell residues, we found further stabilization of native placements and a small but significant (1.7%) number of non‐native placement‐based sites with favorable Rosetta energies, indicating their designability in existing protein interfaces. The generality of the SyPRIS approach allows design of novel symmetric metal sites including with non‐natural amino acid sidechains, and should enable the predictive incorporation of a variety of metal‐containing cofactors at symmetric protein interfaces.     

Modular evolution and the origins of symmetry: reconstruction of a three-fold symmetric globular protein

The high frequency of internal structural symmetry in common protein folds is presumed to reflect their evolutionary origins from the repetition and fusion of ancient peptide modules, but little is known about the primary sequence and physical determinants of this process. Unexpectedly, a sequence and structural analysis of symmetric subdomain modules within an abundant and ancient globular fold, the β-trefoil, reveals that modular evolution is not simply a relic of the ancient past, but is an ongoing and recurring mechanism for regenerating symmetry, having occurred independently in numerous existing β-trefoil proteins. We performed a computational reconstruction of a β-trefoil subdomain module and repeated it to form a newly three-fold symmetric globular protein, ThreeFoil. In addition to its near perfect structural identity between symmetric modules, ThreeFoil is highly soluble, performs multivalent carbohydrate binding, and has remarkably high thermal stability. These findings have far-reaching implications for understanding the evolution and design of proteins via subdomain modules.     

Surface sensing and lateral subcellular localization of WspA,the receptor in a chemosensory‐like system leading to c‐di‐GMP production

Pseudomonas aeruginosa responds to growth on agar surfaces to produce cyclic‐di‐GMP, which stimulates biofilm formation. This is mediated by an alternative cellular function chemotaxis‐like system called Wsp. The receptor protein WspA, is bioinformatically indistinguishable from methyl‐accepting chemotaxis proteins. However, unlike standard chemoreceptors, WspA does not form stable clusters at cell poles. Rather, it forms dynamic clusters at both polar and lateral subcellular locations. To begin to study the mechanism of Wsp signal transduction in response to surfaces, we carried out a structure–function study of WspA and found that its C‐terminus is important for its lateral subcellular localization and function. When this region was replaced with that of a chemoreceptor for amino acids, WspA became polarly localized. In addition, introduction of mutations in the C‐terminal region of WspA that rendered this protein able to form more stable receptor–receptor interactions, also resulted in a WspA protein that was less capable of activating signal transduction. Receptor chimeras with a WspA C‐terminus and N‐terminal periplasmic domains from chemoreceptors that sense amino acids or malate responded to surfaces to produce c‐di‐GMP. Thus, the amino acid sequence of the WspA periplasmic region did not need to be conserved for the Wsp system to respond to surfaces.     

How the folding rates of two‐ and multistate proteins depend on the amino acid properties

Proteins fold by either two‐state or multistate kinetic mechanism. We observe that amino acids play different roles in different mechanism. Many residues that are easy to form regular secondary structures (α helices, β sheets and turns) can promote the two‐state folding reactions of small proteins. Most of hydrophilic residues can speed up the multistate folding reactions of large proteins. Folding rates of large proteins are equally responsive to the flexibility of partial amino acids. Other properties of amino acids (including volume, polarity, accessible surface, exposure degree, isoelectric point, and phase transfer energy) have contributed little to folding kinetics of the proteins. Cysteine is a special residue, it triggers two‐state folding reaction and but inhibits multistate folding reaction. These findings not only provide a new insight into protein structure prediction, but also could be used to direct the point mutations that can change folding rate. Proteins 2014; 82:2375–2382. © 2014 Wiley Periodicals, Inc.     

Hydrophobic clusters in protein structures

Globular proteins fold such that the hydrophobic groups are packed inside forming hydrophobic clusters, and the hydrophilic groups are present on the surface. In this article we analyze clusters of hydrophobic groups of atoms in 781 protein structures selected from the PDB. Our analysis showed that every structure consists of two types of clusters: at least one large cluster that forms the hydrophobic core and probably dictates the protein fold; and numerous smaller clusters, which might be involved in the stabilization of the fold. We also analyzed the preference of the hydrophobic groups in each of the amino acids toward forming hydrophobic clusters. We find that hydrophobic groups from the hydrophilic amino acids also contribute toward cluster formation. Proteins 2008. © 2008 Wiley‐Liss, Inc.     

Detecting local residue environment similarity for recognizing near‐native structure models

We developed a new representation of local amino acid environments in protein structures called the Side‐chain Depth Environment (SDE). An SDE defines a local structural environment of a residue considering the coordinates and the depth of amino acids that locate in the vicinity of the side‐chain centroid of the residue. SDEs are general enough that similar SDEs are found in protein structures with globally different folds. Using SDEs, we developed a procedure called PRESCO (Protein Residue Environment SCOre) for selecting native or near‐native models from a pool of computational models. The procedure searches similar residue environments observed in a query model against a set of representative native protein structures to quantify how native‐like SDEs in the model are. When benchmarked on commonly used computational model datasets, our PRESCO compared favorably with the other existing scoring functions in selecting native and near‐native models. Proteins 2014; 82:3255–3272. © 2014 Wiley Periodicals, Inc.     

A polypeptide "building block" for the β-trefoil fold identified by "top-down symmetric deconstruction"

Fibroblast growth factor-1, a member of the 3-fold symmetric β-trefoil fold, was subjected to a series of symmetric constraint mutations in a process termed “top-down symmetric deconstruction.” The mutations enforced a cumulative exact 3-fold symmetry upon symmetrically equivalent positions within the protein and were combined with a stability screen. This process culminated in a β-trefoil protein with exact 3-fold primary-structure symmetry that exhibited excellent folding and stability properties. Subsequent fragmentation of the repeating primary-structure motif yielded a 42-residue polypeptide capable of spontaneous assembly as a homotrimer, producing a thermostable β-trefoil architecture. The results show that despite pronounced reduction in sequence complexity, pure symmetry in the design of a foldable, thermostable β-trefoil fold is possible. The top-down symmetric deconstruction approach provides a novel alternative means to successfully identify a useful polypeptide “building block” for subsequent “bottom-up” de novo design of target protein architecture.     

How the Sequence of a Gene Specifies Structural Symmetry in Proteins

Internal symmetry is commonly observed in the majority of fundamental protein folds. Meanwhile, sufficient evidence suggests that nascent polypeptide chains of proteins have the potential to start the co-translational folding process and this process allows mRNA to contain additional information on protein structure. In this paper, we study the relationship between gene sequences and protein structures from the viewpoint of symmetry to explore how gene sequences code for structural symmetry in proteins. We found that, for a set of two-fold symmetric proteins from left-handed beta-helix fold, intragenic symmetry always exists in their corresponding gene sequences. Meanwhile, codon usage bias and local mRNA structure might be involved in modulating translation speed for the formation of structural symmetry: a major decrease of local codon usage bias in the middle of the codon sequence can be identified as a common feature; and major or consecutive decreases in local mRNA folding energy near the boundaries of the symmetric substructures can also be observed. The results suggest that gene duplication and fusion may be an evolutionarily conserved process for this protein fold. In addition, the usage of rare codons and the formation of higher order of secondary structure near the boundaries of symmetric substructures might have coevolved as conserved mechanisms to slow down translation elongation and to facilitate effective folding of symmetric substructures. These findings provide valuable insights into our understanding of the mechanisms of translation and its evolution, as well as the design of proteins via symmetric modules.     

Probabilistic approach to the design of symmetric protein quaternary structures

Probabilistic methods have been developed that estimate the site-specific probabilities of the amino acids in sequences likely to fold to a particular target structure, and such information can be used to guide the de novo design of proteins and to probe sequence variability. An extension of these methods for the design of symmetric homo-oligomeric quaternary structures is presented. The theory is in excellent agreement with the results of studies on exactly solvable lattice models. Application to an atomically detailed representation of proteins verifies the utility of a symmetry assumption, which greatly simplifies and accelerates the calculations. The method may be applied to a wide variety of symmetric and periodic protein structures.     

对称蛋白质序列与结构关系研究

进化的观点认为,蛋白质结构的对称性是基因复制和融合的结果,但是由于在长期进化过程中的氨基酸突变,绝大多数现有的蛋白质序列都失去了这种直观的重复性特征。该文简要地回顾了国际上发展的寻找蛋白质序列中重复片段的方法,重点介绍了作者自己提出的分析蛋白质序列和结构对称性的方法以及在蛋白质对称结构形成机理方面的初步工作,并系统分析了各类对称折叠子的序列与结构关系,发现它们的序列都具有隐含的与结构相同的对称性,或者说序列的对称性决定结构的对称性。     

Three reasons protein disorder analysis makes more sense in the light of collagen

We have identified that the collagen helix has the potential to be disruptive to analyses of intrinsically disordered proteins. The collagen helix is an extended fibrous structure that is both promiscuous and repetitive. Whilst its sequence is predicted to be disordered, this type of protein structure is not typically considered as intrinsic disorder. Here, we show that collagen‐encoding proteins skew the distribution of exon lengths in genes. We find that previous results, demonstrating that exons encoding disordered regions are more likely to be symmetric, are due to the abundance of the collagen helix. Other related results, showing increased levels of alternative splicing in disorder‐encoding exons, still hold after considering collagen‐containing proteins. Aside from analyses of exons, we find that the set of proteins that contain collagen significantly alters the amino acid composition of regions predicted as disordered. We conclude that research in this area should be conducted in the light of the collagen helix.     

Amino acid alphabet reduction preserves fold information contained in contact interactions in proteins

To reduce complexity, understand generalized rules of protein folding, and facilitate de novo protein design, the 20‐letter amino acid alphabet is commonly reduced to a smaller alphabet by clustering amino acids based on some measure of similarity. In this work, we seek the optimal alphabet that preserves as much of the structural information found in long‐range (contact) interactions among amino acids in natively‐folded proteins. We employ the Information Maximization Device, based on information theory, to partition the amino acids into well‐defined clusters. Numbering from 2 to 19 groups, these optimal clusters of amino acids, while generated automatically, embody well‐known properties of amino acids such as hydrophobicity/polarity, charge, size, and aromaticity, and are demonstrated to maintain the discriminative power of long‐range interactions with minimal loss of mutual information. Our measurements suggest that reduced alphabets (of less than 10) are able to capture virtually all of the information residing in native contacts and may be sufficient for fold recognition, as demonstrated by extensive threading tests. In an expansive survey of the literature, we observe that alphabets derived from various approaches—including those derived from physicochemical intuition, local structure considerations, and sequence alignments of remote homologs—fare consistently well in preserving contact interaction information, highlighting a convergence in the various factors thought to be relevant to the folding code. Moreover, we find that alphabets commonly used in experimental protein design are nearly optimal and are largely coherent with observations that have arisen in this work. Proteins 2015; 83:2198–2216. © 2015 Wiley Periodicals, Inc.     

A distance‐ and orientation‐dependent energy function of amino acid key blocks

Blocks are the selected portions of amino acids. They have been used effectively to represent amino acids in distinguishing the native conformation from the decoys. Although many statistical energy functions exist, most of them rely on the distances between two or more amino acids. In this study, the authors have developed a pairwise energy function “DOKB” that is both distance and orientation dependent, and it is based on the key blocks that bias the distal ends of side chains. The results suggest that both the distance and the orientation are needed to distinguish the fine details of the packing geometry. DOKB appears to perform well in recognizing native conformations when compared with six other energy functions. Highly packed clusters play important roles in stabilizing the structure. The investigation about the highly packed clusters at the residue level suggests that certain residue pairs in a low‐energy region have lower probability to appear in the highly packed clusters than in the entire protein. The cluster energy term appears to significantly improve the recognition of the native conformations in ig_structal decoy set, in which more highly packed clusters are contained than in other decoy sets. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 681–692, 2014.     

Oligomerization of a symmetric β‐trefoil protein in response to folding nucleus perturbation

Gene duplication and fusion events in protein evolution are postulated to be responsible for the common protein folds exhibiting internal rotational symmetry. Such evolutionary processes can also potentially yield regions of repetitive primary structure. Repetitive primary structure offers the potential for alternative definitions of critical regions, such as the folding nucleus (FN). In principle, more than one instance of the FN potentially enables an alternative folding pathway in the face of a subsequent deleterious mutation. We describe the targeted mutation of the carboxyl‐terminal region of the (internally located) FN of the de novo designed purely‐symmetric β‐trefoil protein Symfoil‐4P. This mutation involves wholesale replacement of a repeating trefoil‐fold motif with a “blade” motif from a β‐propeller protein, and postulated to trap that region of the Symfoil‐4P FN in a nonproductive folding intermediate. The resulting protein (termed “Bladefoil”) is shown to be cooperatively folding, but as a trimeric oligomer. The results illustrate how symmetric protein architectures have potentially diverse folding alternatives available to them, including oligomerization, when preferred pathways are perturbed.     

Three basic regions in adenovirus DNA polymerase interact differentially depending on the protein context to function as bipartite nuclear localization signals.

Adenovirus DNA polymerase (AdPol) contains three clusters of basic amino acids within the N-terminal 48 amino acids: RARR, which begins at amino acid 8, RRRVR, which begins at amino acid 25, and RARRRR, which begins at amino acid 41. These clusters are designated BS I, BS II, and BS III, respectively. (The amino acid codes are: R, arginine; A, alanine; V, valine.) Mutational analysis of these noncontiguous clusters showed that AdPol contains a novel organization of bipartite nuclear localization signals (NLS) that interact differentially to serve in the nuclear targeting of AdPol or of chimeric proteins in which AdPol is linked to Escherichia coli beta-galactosidase (beta-gal). The region containing BS I and BS II functioned interdependently as an NLS for the nuclear targeting of AdPol, for which BS III was dispensible. However, the region containing BS II and BS III constituted a second and more efficient bipartite NLS for the nuclear targeting of the AdPol-E. coli beta-gal fusion protein. Moreover, deletion or limited insertion of amino acids in the spacer region between BS II and BS III did not affect their nuclear targeting function for these fusion proteins. Chou-Fasman predictive analysis of protein secondary structure in the vicinity of the bipartite NLS sequences supports a model in which protein conformation in the spacer region may play an important role in bringing these clusters of basic amino acids into close proximity, allowing them to function as nuclear targeting signals for this class of nuclear proteins.     

Comparative characterization of random‐sequence proteins consisting of 5, 12, and 20 kinds of amino acids

Screening of functional proteins from a random‐sequence library has been used to evolve novel proteins in the field of evolutionary protein engineering. However, random‐sequence proteins consisting of the 20 natural amino acids tend to aggregate, and the occurrence rate of functional proteins in a random‐sequence library is low. From the viewpoint of the origin of life, it has been proposed that primordial proteins consisted of a limited set of amino acids that could have been abundantly formed early during chemical evolution. We have previously found that members of a random‐sequence protein library constructed with five primitive amino acids show high solubility (Doi et al., Protein Eng Des Sel 2005;18:279–284). Although such a library is expected to be appropriate for finding functional proteins, the functionality may be limited, because they have no positively charged amino acid. Here, we constructed three libraries of 120‐amino acid, random‐sequence proteins using alphabets of 5, 12, and 20 amino acids by preselection using mRNA display (to eliminate sequences containing stop codons and frameshifts) and characterized and compared the structural properties of random‐sequence proteins arbitrarily chosen from these libraries. We found that random‐sequence proteins constructed with the 12‐member alphabet (including five primitive amino acids and positively charged amino acids) have higher solubility than those constructed with the 20‐member alphabet, though other biophysical properties are very similar in the two libraries. Thus, a library of moderate complexity constructed from 12 amino acids may be a more appropriate resource for functional screening than one constructed from 20 amino acids.     

Structural diversity of sequentially identical subsequences of proteins: Identical octapeptides can have different conformations

One of the most important questions in the protein folding problem is whether secondary structures are formed entirely by local interactions. One way to answer this question is to compare identical subsequences of proteins to see if they have identical structures. Such an exercise would also reveal a lower limit on the number of amino acids needed to form unique secondary structures. In this context, we have searched the April 1996 release of the Protein Data Bank for sequentially identical subsequences of proteins and compared their structures. We find that identical octamers can have different conformations. In addition, there are several examples of identical heptamers with different conformations, and the number of identical hexamers with different conformations has increased since the previous PDB releases. These observations imply that secondary structure can be formed entirely by non-local interactions and that an identical match of up to eight amino acids may not imply structural similarity. In addition to the larger context of the protein folding problem, these observations have implications for protein structure prediction methods. Proteins 30:228–231, 1998. © 1998 Wiley-Liss, Inc.     

Design and structure of two new protein cages illustrate successes and ongoing challenges in protein engineering

In recent years, new protein engineering methods have produced more than a dozen symmetric, self‐assembling protein cages whose structures have been validated to match their design models with near‐atomic accuracy. However, many protein cage designs that are tested in the lab do not form the desired assembly, and improving the success rate of design has been a point of recent emphasis. Here we present two protein structures solved by X‐ray crystallography of designed protein oligomers that form two‐component cages with tetrahedral symmetry. To improve on the past tendency toward poorly soluble protein, we used a computational protocol that favors the formation of hydrogen‐bonding networks over exclusively hydrophobic interactions to stabilize the designed protein–protein interfaces. Preliminary characterization showed highly soluble expression, and solution studies indicated successful cage formation by both designed proteins. For one of the designs, a crystal structure confirmed at high resolution that the intended tetrahedral cage was formed, though several flipped amino acid side chain rotamers resulted in an interface that deviates from the precise hydrogen‐bonding pattern that was intended. A structure of the other designed cage showed that, under the conditions where crystals were obtained, a noncage structure was formed wherein a porous 3D protein network in space group I2₁3 is generated by an off‐target twofold homomeric interface. These results illustrate some of the ongoing challenges of developing computational methods for polar interface design, and add two potentially valuable new entries to the growing list of engineered protein materials for downstream applications.