Evidence for specific subunit distribution and interactions in the quaternary structure of α‐crystallin

The quaternary structure of α‐crystallin is dynamic, a property which has thwarted crystallographic efforts towards structural characterization. In this study, we have used collision‐induced dissociation mass spectrometry to examine the architecture of the polydisperse assemblies of α‐crystallin. For total α‐crystallin isolated directly from fetal calf lens using size‐based chromatography, the αB‐crystallin subunit was found to be preferentially dissociated from the oligomers, despite being significantly less abundant overall than the αA‐crystallin subunits. Furthermore, upon mixing molar equivalents of purified αA‐ and αB‐crystallin, the levels of their dissociation were found to decrease and increase, respectively, with time. Interestingly though, dissociation of subunits from the αA‐ and αB‐crystallin homo‐oligomers was comparable, indicating that strength of the αA:αA, and αB:αB subunit interactions are similar. Taken together, these data suggest that the differences in the number of subunit contacts in the mixed assemblies give rise to the disproportionate dissociation of αB‐crystallin subunits. Limited proteolysis mass spectrometry was also used to examine changes in protease accessibility during subunit exchange. The C‐terminus of αA‐crystallin was more susceptible to proteolytic attack in homo‐oligomers than that of αB‐crystallin. As subunit exchange proceeded, proteolysis of the αA‐crystallin C‐terminus increased, indicating that in the hetero‐oligomeric form this tertiary motif is more exposed to solvent. These data were used to propose a refined arrangement for the interactions of the α‐crystallin domains and C‐terminal extensions of subunits within the α‐crystallin assembly. In particular, we propose that the palindromic IPI motif of αB‐crystallin gives rise to two orientations of the C‐terminus. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Cyclic oligomer design with de novo αβ‐proteins

We have previously shown that monomeric globular αβ‐proteins can be designed de novo with considerable control over topology, size, and shape. In this paper, we investigate the design of cyclic homo‐oligomers from these starting points. We experimented with both keeping the original monomer backbones fixed during the cyclic docking and design process, and allowing the backbone of the monomer to conform to that of adjacent subunits in the homo‐oligomer. The latter flexible backbone protocol generated designs with shape complementarity approaching that of native homo‐oligomers, but experimental characterization showed that the fixed backbone designs were more stable and less aggregation prone. Designed C2 oligomers with β‐strand backbone interactions were structurally confirmed through x‐ray crystallography and small‐angle X‐ray scattering (SAXS). In contrast, C3‐C5 designed homo‐oligomers with primarily nonpolar residues at interfaces all formed a range of oligomeric states. Taken together, our results suggest that for homo‐oligomers formed from globular building blocks, improved structural specificity will be better achieved using monomers with increased shape complementarity and with more polar interfaces.     

Conservation of Functionally Important Global Motions in an Enzyme Superfamily across Varying Quaternary Structures

The α‐d‐phosphohexomutase superfamily comprises enzymes involved in carbohydrate metabolism that are found in all kingdoms of life. Recent biophysical studies have shown for the first time that several of these enzymes exist as dimers in solution, prompting an examination of the oligomeric state of all proteins of known structure in the superfamily (11 different proteins; 31 crystal structures) via computational and experimental analyses. We find that these proteins range in quaternary structure from monomers to tetramers, with 6 of the 11 known structures being likely oligomers. The oligomeric state of these proteins not only is associated in some cases with enzyme subgroup (i.e., substrate specificity) but also appears to depend on domain of life, with the two archaeal proteins existing as higher‐order oligomers. Within the oligomers, three distinct interfaces are observed, one of which is found in both archaeal and bacterial proteins. Normal mode analysis shows that the topological arrangement of the oligomers permits domain 4 of each protomer to move independently as required for catalysis. Our analysis suggests that the advantages associated with protein flexibility in this enzyme family are of sufficient importance to be maintained during the evolution of multiple independent oligomers. This study is one of the first showing that global motions may be conserved not only within protein families but also across members of a superfamily with varying oligomeric structures.     

Structural templates for modeling homodimers

Oligomeric proteins are more abundant in nature than monomeric proteins, and involved in all biological processes. In the absence of an experimental structure, their subunits can be modeled from their sequence like monomeric proteins, but reliable procedures to build the oligomeric assembly are scarce. Template‐based methods, which start from known protein structures, are commonly applied to model subunits. We present a method to model homodimers that relies on a structural alignment of the subunits, and test it on a set of 511 target structures recently released by the Protein Data Bank, taking as templates the earlier released structures of 3108 homodimeric proteins (H‐set), and 2691 monomeric proteins that form dimer‐like assemblies in crystals (M‐set). The structural alignment identifies a H‐set template for 97% of the targets, and in half of the cases, it yields a correct model of the dimer geometry and residue–residue contacts in the target. It also identifies a M‐set template for most of the targets, and some of the crystal dimers are very similar to the target homodimers. The procedure efficiently detects homology at low levels of sequence identities, and points to erroneous quaternary structures in the Protein Data Bank. The high coverage of the target set suggests that the content of the Protein Data Bank already approaches the structural diversity of protein assemblies in nature, and that template‐based methods should become the choice method for modeling oligomeric as well as monomeric proteins.     

The TET2 and TET3 aminopeptidases from Pyrococcus horikoshii form a hetero‐subunit peptidasome with enhanced peptide destruction properties

TET aminopeptidases assemble as large homo‐dodecameric complexes. The reason why prokaryotic genomes often encode a diverse set of TET peptidases homologues remains unclear. In the archaeon Pyrococcus horikoshii, PhTET1, PhTET2 and PhTET3 homo‐oligomeric particles have been proposed to work in concert to breakdown intracellular polypeptides. When coexpressed in Escherichia coli, the PhTET2 and PhTET3 proteins were found to assemble efficiently as heteromeric complexes. Biophysical analysis demonstrated that these particles possess the same quaternary structure as the homomeric TET dodecamers. The same hetero‐oligomeric complexes were immunodetected in P. horikoshii cell extracts analysed by sucrose gradient fractionation and ion exchange chromatography. The biochemical activity of a purified hetero‐oligomeric TET particle, assessed on chromogenic substrates and on a complex mixture of peptides, reveals that it displays higher efficiency than an equivalent combination of homo‐oligomeric TET particles. Interestingly, phylogenetic analysis shows that PhTET2 and PhTET3 are paralogous proteins that arose from gene duplication in the ancestor of Thermococcales. Together, these results establish that the PhTET2 and PhTET3 proteins are two subunits of the same enzymatic complex aimed at the destruction of polypeptidic chains of very different composition. This is the first report for such a mechanism intended to improve multi‐enzymatic complex efficiency among exopeptidases.     

αB-crystallin polydispersity is a consequence of unbiased quaternary dynamics

The inherent heterogeneity of many protein assemblies complicates characterization of their structure and dynamics, as most biophysical techniques require homogeneous preparations of isolated components. For this reason, quantitative studies of the molecular chaperone αB-crystallin, which populates a range of interconverting oligomeric states, have been difficult, and the physicochemical basis for its polydispersity has remained unknown. Here, we perform mass spectrometry experiments to study αB-crystallin and extract detailed information as to its oligomeric distribution and exchange of subunits under a range of conditions. This allows a determination of the thermodynamic and kinetic parameters that govern the polydisperse ensemble and enables the construction of a simple energy profile for oligomerization. We find that the quaternary structure and dynamics of the protein can be explained using a simple model with just two oligomer-independent interactions (i.e., interactions that are energetically identical in all oligomers from 10mers to 40mers) between constituent monomers. As such, the distribution of oligomers is governed purely by the dynamics of individual monomers. This provides a new means for understanding the polydispersity of αB-crystallin and a framework for interrogating other heterogeneous protein assemblies.     

Surface recognition elements of membrane protein oligomerization

Although certain membrane proteins are functional as monomeric polypeptides, others must assemble into oligomers to carry out their biological roles. High-resolution membrane protein structures provide a valuable resource for examining the sequence features that facilitate-or preclude-assembly of membrane protein monomers into multimeric structures. Here we have utilized a data set of 28 high-resolution alpha-helical membrane protein structures comprising 32 nonredundant polypeptides to address this issue. The lipid-exposed surfaces of membrane proteins that have reached their fully assembled and functional biological units have been compared with those of the individual subunits that build quaternary structures. Though the overall amino acid composition of each set of surfaces is similar, a key distinction-the distribution of small-xxx-small motifs-delineates subunits from membrane proteins that have reached a functioning oligomeric state. Quaternary structure formation may therefore be dictated by small-xxx-small motifs that are not satisfied by intrachain contacts.     

Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states

Fucoxanthin-chlorophyll proteins were purified from the centric diatom Cyclotella meneghiniana. Two major fractions were observed that differed in their polypeptide composition and oligomeric state. Trimers consist of mainly 18 kDa polypeptides. Higher oligomers are tightly assembled from different trimers, which contain mostly 19 kDa subunits. In both oligomeric states, the excitation energy coupling between fucoxanthin and chlorophyll a was preserved, and chlorophyll c was shown to transfer energy efficiently to chlorophyll a. Circular dichroism spectra showed close interaction between fucoxanthin and chlorophyll a, and different chlorophyll a molecules were demonstrated to interact excitonically. The assembly of trimers of antenna proteins with a distinct subunit composition into higher oligomeric states was not reported so far and differs from the situation found in higher plants. The differences in the supramolecular structure of the fucoxanthin-chlorophyll proteins reflect the dissimilar arrangement of the thylakoid membranes in diatoms, which lack the grana-stroma distinction.     

Amino acid substitutions at protein–protein interfaces that modulate the oligomeric state

Despite similarities in their sequence and structure, there are a number of homologous proteins that adopt various oligomeric states. Comparisons of these homologous protein pairs, in terms of residue substitutions at the protein–protein interfaces, have provided fundamental characteristics that describe how proteins interact with each other. We have prepared a dataset composed of pairs of related proteins with different homo‐oligomeric states. Using the protein complexes, the interface residues were identified, and using structural alignments, the shadow‐interface residues have been defined as the surface residues that align with the interface residues. Subsequently, we investigated residue substitutions between the interfaces and the shadow interfaces. Based on the degree of the contributions to the interactions, the aligned sites of the interfaces and shadow interfaces were divided into primary and secondary sites; the primary sites are the focus of this work. The primary sites were further classified into two groups (i.e. exposed and buried) based on the degree to which the residue is buried within the shadow interfaces. Using these classifications, two simple mechanisms that mediate the oligomeric states were identified. In the primary‐exposed sites, the residues on the shadow interfaces are replaced by more hydrophobic or aromatic residues, which are physicochemically favored at protein–protein interfaces. In the primary‐buried sites, the residues on the shadow interfaces are replaced by larger residues that protrude into other proteins. These simple rules are satisfied in 23 out of 25 Structural Classification of Proteins (SCOP) families with a different‐oligomeric‐state pair, and thus represent a basic strategy for modulating protein associations and dissociations. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Quaternary structure of alpha-crystallin is necessary for the binding of unfolded proteins: a surface plasmon resonance study

The interactions between an oligomeric heat-shock protein, alpha-crystallin, and its individual subunits with unfolded proteins were monitored by surface plasmon resonance. Immobilization at the sensor chip allowed us for the first time to study isolated alpha-crystallin subunits under physiological conditions. We observe that these subunits, in contrast to alpha-crystallin oligomers, do not bind unfolded protein. Our data indicate that quaternary structure of alpha-crystallin is necessary for its chaperone-like activity.     

The crystal structure of the Calystegia sepium agglutinin reveals a novel quaternary arrangement of lectin subunits with a beta-prism fold

The high number of quaternary structures observed for lectins highlights the important role of these oligomeric assemblies during carbohydrate recognition events. Although a large diversity in the mode of association of lectin subunits is frequently observed, the oligomeric assemblies of plant lectins display small variations within a single family. The crystal structure of the mannose-binding jacalin-related lectin from Calystegia sepium (Calsepa) has been determined at 1.37-A resolution. Calsepa exhibits the same beta-prism fold as identified previously for other members of the family, but the shape and the hydrophobic character of its carbohydrate-binding site is unlike that of other members, consistent with surface plasmon resonance analysis showing a preference for methylated sugars. Calsepa reveals a novel dimeric assembly markedly dissimilar to those described earlier for Heltuba and jacalin but mimics the canonical 12-stranded beta-sandwich dimer found in legume lectins. The present structure exemplifies the adaptability of the beta-prism building block in the evolution of plant lectins and highlights the biological role of these quaternary structures for carbohydrate recognition.     

Dimer structure and conformational variability in the N-terminal region of an archaeal small heat shock protein, StHsp14.0

Small heat shock proteins (sHsps), which are categorized into a class of molecular chaperones, bind and stabilize denatured proteins to prevent aggregation. The sHsps undergo transition between different oligomeric states to control their hydrophobicity. So far, only the structures of sHsps in large oligomeric states have been reported. Here we report the structure of StHsp14.0 from Sulfolobus tokodaii in the dimeric state, which is formed by means of a mutation at the C-terminal IXI/V motif. The dimer is the sole building block in two crystal forms, and the dimeric mode is the same as that in the large oligomers. The N-terminal helix has variety in its conformation. Furthermore, spectroscopic and biochemical experiments were performed to investigate the conformational variability at the N-terminus. The structural, dynamical and oligomeric properties suggest that chaperone activity of StHsp14.0 is mediated by partially dissolved oligomers.     

Incomplete pneumolysin oligomers form membrane pores

Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.     

Structural studies on the oligomeric transition of a small heat shock protein, StHsp14.0

The small heat shock proteins (sHsps), which are widely found in all domains of life, bind and stabilize denatured proteins to prevent aggregation. The sHsps exist as large oligomers that are composed of 9–40 subunits and control their chaperone activity by the transition of the oligomeric state. Though the oligomeric transition is important for the biological function of most sHsps, atomic details have not been elucidated. Here, we report crystal structures in both the 24-meric and dimeric states for an sHsp, StHsp14.0 from Sulfolobus tokodaii, in order to reveal changes upon the oligomeric transition. The results indicate that StHsp14.0 forms a spherical 24-mer with a diameter of 115 Å. The diameter is defined by the inter-monomer angle in the dimer. The dimer structure in the dimeric state shows only small differences from that in the 24-meric state. Some significant differences are exclusively observed at the binding site for the C-terminus. Although a dimer has four interactive sites with neighboring dimers, the weakness of the respective interactions is indicated from the size-exclusion chromatography. The small structural changes imply an activation mechanism mediated by multiple weak interactions.     

Quaternary structure constraints on evolutionary sequence divergence

The structurally constrained protein evolution (SCPE) model simulates protein divergence considering protein structure explicitly. The model is based on the observation that protein structure is more conserved during evolution than the sequences encoding for that structure. In the previous work, the SCPE model considered only the tertiary structure. Here we show that the performance of the model is enhanced when the oligomeric structure is taken into account. Our results agree with recent evolutionary studies of oligomeric proteins, which show that conservation of the quaternary structure imposes additional constraints on sequence divergence. The incorporation of protein-protein interactions into protein evolution models may be important in the study of quaternary protein structures and complex protein assemblies.     

The Volumetric Diversity of Misfolded Prion Protein Oligomers Revealed by Pressure Dissociation

Protein oligomerization has been associated with a wide range of diseases. High pressure approaches offer a powerful tool for deciphering the underlying molecular mechanisms by revealing volume changes associated with the misfolding and assembly reactions. We applied high pressure to induce conformational changes in three distinct β-sheet-rich oligomers of the prion protein PrP, a protein characterized by a variety of infectious quaternary structures that can propagate stably and faithfully and cause diseases with specific phenotypic traits. We show that pressure induces dissociation of the oligomers and leads to a lower volume monomeric PrP state that refolds into the native conformation after pressure release. By measuring the different pressure and temperature sensitivity of the tested PrP oligomers, we demonstrate significantly different void volumes in their quaternary structure. In addition, by focusing on the kinetic and energetic behavior of the pressure-induced dissociation of one specific PrP oligomer, we reveal a large negative activation volume and an increase in both apparent activation enthalpy and entropy. This suggests a transition state ensemble that is less structured and significantly more hydrated than the oligomeric state. Finally, we found that site-specific fluorescent labeling allows monitoring of the transient population of a kinetic intermediate in the dissociation reaction. Our results indicate that defects in atomic packing may deserve consideration as a new factor that influences differences between PrP assemblies and that could be relevant also for explaining the origin of prion strains.     

Self-assembly of biological macromolecules.

The genetic apparatus of the cell is responsible for the accurate biosynthesis of the primary structure of macromolecules which then spontaneously fold up and, in certain circumstances, aggregate to yield the complex tertiary and quaternary structures of the biologically active molecules. Structures capable of self-assembly in this range from simple monomers through oligomers to complex multimeric structures that may contain more than one type of polypeptide chain and components other than protein. It is becoming clear that even with the simpler monomeric enzymes there is becoming clear that even with the simpler monomeric enzymes there is a kinetically determined pathway for the folding process and that a folded protein must now be regarded as the minimum free energy form of the kinetically accessible conformations. It is argued that the denatured subunits of oligomeric enzymes are likely to fold to something like their final structure before aggregating to give the native quaternary structure and the available evidence would suggest that this is so. The importance of nucleation events and stable intermediates in the self-assembly of more complex structures is clear. Many self-assembling structures contain only identical subunits and symmetry arguments are very successful in accounting for the structures formed. Because proteins are themselves complex molecules and not inelastic geometric objects, the rules of strict symmetry can be bent and quasi-equivalent bonding between subunits permitted. This possibility is frequently employed in biological structures. Conversely, symmetry arguments can offer a reliable means of choosing between alternative models for a given structure. It can be seen that proteins gain stability by growing larger and it is argued in evolutionary terms that aggregation of subunits is the preferred way to increase the size of proteins. The possession of quaternary structure by enzymes allows conferral of other biologically important properties, such as cooperativity between active sites, changes of specificity, substrate channelling and sequential reactions within a multi-enzyme complex. Comparison is made of the invariant subunit compositions of the simpler oligomeric enzymes with the variation evidently open to, say, the 2-oxoacid dehydrogenase complexes of E. coli. With viruses, on the other hand, the function of the quaternary structure is to package nucleic acid and, as an example, the assembly and breakdown of tobacco mosaic virus is discussed. Attention is drawn to the possible ways in which the principles of self-assembly can be extended to make structures more complicated than those that can be formed by simple aggregation of the comonent parts.     

Large oligomeric complex structures can be computationally assembled by efficiently combining docked interfaces

Macromolecular oligomeric assemblies are involved in many biochemical processes of living organisms. The benefits of such assemblies in crowded cellular environments include increased reaction rates, efficient feedback regulation, cooperativity and protective functions. However, an atom‐level structural determination of large assemblies is challenging due to the size of the complex and the difference in binding affinities of the involved proteins. In this study, we propose a novel combinatorial greedy algorithm for assembling large oligomeric complexes from information on the approximate position of interaction interfaces of pairs of monomers in the complex. Prior information on complex symmetry is not required but rather the symmetry is inferred during assembly. We implement an efficient geometric score, the transformation match score, that bypasses the model ranking problems of state‐of‐the‐art scoring functions by scoring the similarity between the inferred dimers of the same monomer simultaneously with different binding partners in a (sub)complex with a set of pregenerated docking poses. We compiled a diverse benchmark set of 308 homo and heteromeric complexes containing 6 to 60 monomers. To explore the applicability of the method, we considered 48 sets of parameters and selected those three sets of parameters, for which the algorithm can correctly reconstruct the maximum number, namely 252 complexes (81.8%) in, at least one of the respective three runs. The crossvalidation coverage, that is, the mean fraction of correctly reconstructed benchmark complexes during crossvalidation, was 78.1%, which demonstrates the ability of the presented method to correctly reconstruct topology of a large variety of biological complexes. Proteins 2015; 83:1887–1899. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Conformational changes in redox pairs of protein structures

Disulfides are conventionally viewed as structurally stabilizing elements in proteins but emerging evidence suggests two disulfide subproteomes exist. One group mediates the well known role of structural stabilization. A second redox‐active group are best known for their catalytic functions but are increasingly being recognized for their roles in regulation of protein function. Redox‐active disulfides are, by their very nature, more susceptible to reduction than structural disulfides; and conversely, the Cys pairs that form them are more susceptible to oxidation. In this study, we searched for potentially redox‐active Cys Pairs by scanning the Protein Data Bank for structures of proteins in alternate redox states. The PDB contains over 1134 unique redox pairs of proteins, many of which exhibit conformational differences between alternate redox states. Several classes of structural changes were observed, proteins that exhibit: disulfide oxidation following expulsion of metals such as zinc; major reorganisation of the polypeptide backbone in association with disulfide redox‐activity; order/disorder transitions; and changes in quaternary structure. Based on evidence gathered supporting disulfide redox activity, we propose disulfides present in alternate redox states are likely to have physiologically relevant redox activity.