Homo-oligomeric Proteins Research Articles

Bacterial microcompartment shells of diverse functional types possess pentameric vertex proteins

Bacterial microcompartments (MCPs) are large proteinaceous structures comprised of a roughly icosahedral shell and a series of encapsulated enzymes. MCPs carrying out three different metabolic functions have been characterized in some detail, while gene expression and bioinformatics studies have implicated other types, including one believed to perform glycyl radical-based metabolism of 1,2-propanediol (Grp). Here we report the crystal structure of a protein (GrpN), which is presumed to be part of the shell of a Grp-type MCP in Rhodospirillum rubrum F11. GrpN is homologous to a family of proteins (EutN/PduN/CcmL/CsoS4) whose members have been implicated in forming the vertices of MCP shells. Consistent with that notion, the crystal structure of GrpN revealed a pentameric assembly. That observation revived an outstanding question about the oligomeric state of this protein family: pentameric forms (for CcmL and CsoS4A) and a hexameric form (for EutN) had both been observed in previous crystal structures. To clarify these confounding observations, we revisited the case of EutN. We developed a molecular biology-based method for accurately determining the number of subunits in homo-oligomeric proteins, and found unequivocally that EutN is a pentamer in solution. Based on these convergent findings, we propose the name bacterial microcompartment vertex for this special family of MCP shell proteins.

Intertwined Associations in Structures of Homooligomeric Proteins

Intertwined homo-oligomers are complexes comprising identical protein subunits, where small segments or compact protein substructures (domains) are exchanged between the subunits. Using a formal definition of intertwined homo-oligomers, we survey the Protein Data Bank for all such complexes. Results show that intertwining occurs in 13,442 (24%) of all surveyed structures. A majority (∼72%) exchanges one contiguous chain segment of varyinglength. Another ∼10%, exchange structural domains, and the remaining ∼20% display complex intertwining topologies. Smaller proteins are more often intertwined, and intertwining is dominant in solution homodimers. These findings and analyses of various properties of the major category of intertwined complexes, their interfaces and quaternary context, support the physiological role of intertwining in promoting homooligomer stability. Furthermore, the number of different intertwining modes observed in families of related proteins is limited, and likely specific to the protein fold. These findings yield unique insights into the role of intertwining in homomeric association.

Protein β-interfaces as a generic source of native peptide tectons

Motifs of 7-8 amino acids were designed from the β-continuous interfaces of non-related homo-oligomeric proteins. These peptides intrinsically self-assembled into nanoarchitectures in water, while retaining some properties of their parent interfaces, especially reversibility of assembly. These results reveal a novel source of native peptide tectons.

Influence of Structural Symmetry on Protein Dynamics

Structural symmetry in homooligomeric proteins has intrigued many researchers over the past several decades. However, the implication of protein symmetry is still not well understood. In this study, we performed molecular dynamics (MD) simulations of two forms of trp RNA binding attenuation protein (TRAP), the wild-type 11-mer and an engineered 12-mer, having two different levels of circular symmetry. The results of the simulations showed that the inter-subunit fluctuations in the 11-mer TRAP were significantly smaller than the fluctuations in the 12-mer TRAP while the internal fluctuations were larger in the 11-mer than in the 12-mer. These differences in thermal fluctuations were interpreted by normal mode analysis and group theory. For the 12-mer TRAP, the wave nodes of the normal modes existed at the flexible interface between the subunits, while the 11-mer TRAP had its nodes within the subunits. The principal components derived from the MD simulations showed similar mode structures. These results demonstrated that the structural symmetry was an important determinant of protein dynamics in circularly symmetric homooligomeric proteins.

Blind prediction of quaternary structures of homo-oligomeric proteins from amino acid sequences based on templates

Background: Prediction of protein tertiary and quaternary structures helps us to understand protein functionality. While tertiary structure prediction techniques have been much improved over the last two

Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study

The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate- (AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.

Kinetic Asymmetry of Subunit Exchange of Homooligomeric Protein as Revealed by Deuteration-Assisted Small-Angle Neutron Scattering

We developed a novel, to our knowledge, technique for real-time monitoring of subunit exchange in homooligomeric proteins, using deuteration-assisted small-angle neutron scattering (SANS), and applied it to the tetradecamer of the proteasome α7 subunit. Isotopically normal and deuterated tetradecamers exhibited identical SANS profiles in 81% D2O solution. After mixing these solutions, the isotope sensitive SANS intensity in the low-q region gradually decreased, indicating subunit exchange, whereas the small-angle x-ray scattering profile remained unchanged confirming the structural integrity of the tetradecamer particles during the exchange. Kinetic analysis of zero-angle scattering intensity indicated that 1), only two of the 14 subunits were exchanged in each tetradecamer and 2), the exchange process involves at least two steps. This study underscores the usefulness of deuteration-assisted SANS, which can provide quantitative information not only on the molecular sizes and shapes of homooligomeric proteins, but also on their kinetic properties.

OligoPred: A web-server for predicting homo-oligomeric proteins by incorporating discrete wavelet transform into Chou's pseudo amino acid composition

In vivo, some proteins exist as monomers (single polypeptide chains) and others as oligomers. Not like monomers, oligomers are composed of two or more chains (subunits) that are associated with each other through non-covalent interactions and, occasionally, through disulfide bonds. These proteins are the structural components of various biological functions, including cooperative effects, allosteric mechanisms and ion-channel gating. However, with the dramatic increase in the number of protein sequences submitted to the public data bank, it is important for both basic research and drug discovery research to acquire the possible knowledge about homo-oligomeric attributes of their interested proteins in a timely manner. In this paper, a high-throughput method, combined support vector machines with discrete wavelet transform, has been developed to predict the protein homo-oligomers. The total accuracy obtained by the re-substitution test, jackknife test and independent dataset test are 99.94%, 96.17% and 96.18%, respectively, showing that the proposed method of extracting feature from the protein sequences is effective and feasible for predicting homo-oligomers. The online service is available at http://bioinfo.ncu.edu.cn/Services.aspx.

Morpheeins – A New Pathway For Allosteric Drug Discovery~!2010-02-12~!2010-05-21~!2010-06-08~!

The morpheein model of allosteric regulation can be applied as a novel approach to the discovery of small molecule allosteric modulators of protein function. Morpheeins are homo-oligomeric proteins where, under physiological conditions, the oligomer can dissociate, the dissociated units can change conformation, and the altered conformational state can reassociate to a structurally and functionally distinct oligomer. This phenomenon serves as a basis for allostery, as a basis for conformational diseases, as a basis for drug discovery, and may be applicable to personalized medicine such as in the prediction of drug side effects. Each of these relationships has been established for the prototype morpheein, porphobilinogen synthase, where the conformational disease is a porphyria and the drug application is in antimicrobial discovery. These data are presented along with a discussion of other drug targets for which the morpheein model of allostery may apply. Such targets include HIV integrase, TNFα, β-tryptase, and p53.

Protein quaternary structure focus for small molecule allosteric control

A subset of homo‐oligomeric proteins can reversibly come apart, change conformation in the dissociated state, and reassemble into a structurally and functionally distinct oligomer. Work with the enzyme porphobilinogen synthase has established that this sort of quaternary structure dynamic is physiologically relevant. Proteins that can rearrange in this way are called morpheeins and their quaternary structure dynamic impacts our understanding of protein function and small molecule protein interactions in the following ways. 1) The quaternary structure dynamic of morpheeins can form a structural basis for allostery. 2) Small molecule stabilization of one of the alternate quaternary assemblies defines a new approach to allosteric drug discovery. 3) In addition to functioning as drugs, small molecules that bind to oligomer specific surface cavities will alter the quaternary structure equilibrium and can cause off‐target drug side effects. 4) Naturally occurring mutations can alter the normal quaternary structure equilibrium. This alters normal function and can contribute to disease states. We describe how each of these four impacts has been demonstrated with the morpheein porphobilinogen synthase. We propose additional medically relevant proteins whose behavior suggests that they may function as morpheeins.

Modular Control of Cross-oligomerization: ANALYSIS OF SUPERSTABILIZED Hsp90 HOMODIMERS IN VIVO

Homo-oligomeric proteins fulfill numerous functions in all cells. The ability to co-express subunits of these proteins that preferentially self-assemble without cross-oligomerizing provides for controlled experiments to analyze the function of mutant homo-oligomers in vivo. Hsp90 is a dimeric chaperone involved in the maturation of many kinases and steroid hormone receptors. We observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered superstabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. Superstabilized Hsp90 dimers supported robust growth of S. cerevisiae, indicating that dissociation of Hsp90 dimers could be hindered without compromising essential function. We utilized superstabilized dimers to analyze the activity of ATPase mutant homodimers in a temperature-sensitive yeast background where elevated temperature inactivated all other Hsp90 species. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of glucocorticoid receptor and v-Src, confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo.

Mimicking the evolution of a thermally stable monomeric four-helix bundle by fusion of four identical single-helix peptides

Internal symmetry is a common feature of the tertiary structures of proteins and protein domains. Probably, because the genes of homo-oligomeric proteins duplicated and fused, their evolutionary descendants are proteins with internal symmetry. To identify any advantages that cause monomeric proteins with internal symmetry to be selected evolutionarily, we characterized some of the physical properties of a recombinant protein with a sequence consisting of two tandemly fused copies of the Escherichia coli Lac repressor C-terminal alpha-helix. This polypeptide exists in solution mainly as dimer that likely maintains a four-helix bundle motif. Thermal unfolding experiments demonstrate that the protein is considerably more stable at elevated temperatures than is a homotetramer consisting of four non-covalently associated copies of a 21-residue polypeptide similar in sequence to that of the Lac repressor C-terminal alpha-helix. A tandem duplication of our helix-loop-helix polypeptide yields an even more thermally stable protein. Our results exemplify the concept that fusion of non-covalently assembled polypeptide chains leads to enhanced protein stability. Herein, we discuss how our work relates to the evolutionary selective-advantages realized when symmetrical homo-oligomers evolve into monomers. Moreover, our thermally stable single-chain four-helix bundle protein may provide a robust scaffold for development of new biomaterials.

The Dominance of Symmetry in the Evolution of Homo-oligomeric Proteins

The fact that aggregates of identical protein molecules are usually symmetric has remained an enigma. An idealized model of a soluble monomeric protein was constructed and accompanied through a simulated evolutionary process resulting in dimerization, in order to elucidate this peculiarity. The model showed that the probability of a symmetric association is by a factor of 100 or above higher than the probability of an asymmetric one. Unexpectedly, symmetry prevails in the dimer initiation phase much more than in the dimer improvement phase of evolution. The result is clear-cut and robust against a broad spectrum of model inadequacies. It rationalizes the predominance of symmetric homo-oligomers.

Controlling oligomerization through protein engineering: in vivo analysis of Hsp90

Many homo-oligomeric proteins are vital for biology including ion-channels, the p53 tumor suppressor, and the essential kinase-associated chaperone Hsp90. Mutational analyses of these homo-oligomeric systems in vivo is complicated by cross-oligomerization between wild-type and mutant subunits. We have devised a generalizable thermodynamic strategy to prevent cross-dimerization. Appending an oligomerization domain to the mutant subunits reduces the free energy of homocomplexes relative to wild-type/mutant heterocomplexes. We have used this strategy to engineer super-stabilized Hsp90 dimers that do not cross-oligomerize with wild-type Hsp90. Super-stabilized Hsp90 supports yeast viability and is fully active in the maturation of v-src kinase. Thus, our stabilization strategy does not disturb the biochemical function of Hsp90.We have used superstabilized Hsp90 to address a fundamental and long-unanswered question regarding Hsp90: what clients or substrate proteins depend on Hsp90 ATPase activity in vivo. The identification of ATP dependent Hsp90 substrates has been a major challenge both in vitro and in vivo. In vitro studies are complicated by the large number of co-chaperones required for Hsp90 to function efficiently. In vivo studies are complicated both because ATPase deficient Hsp90 mutants do not support viability and because when different Hsp90 variants are co-expressed they form a mixture of different dimer species. We have used our engineered super-stabilized Hsp90 to developed a yeast system to identify clients that rely on Hsp90 ATPase activity. Using this approach, we find that Hsp90 mutants deficient for ATP binding or hydrolysis have differential impacts on the activation of kinase and hormone receptor clients in vivo. These results provide a rationale for understanding anti-cancer drugs that competitively bind to the ATPase site of Hsp90.

Is protein structure prediction still an enigma

Proteins are large molecules indispensable for the existence and proper functioning of biological organisms. They perform a wide array of functions including catalysis, structure formation, transport, body defense, etc. Understanding the functions of proteins is a fundamental problem in the discovery of drugs to treat various diseases. The structure of a protein can be determined by physical methods which are slow and expensive but owing to the dramatic increase in the numbers of proteins sent to the public data bank during the last few years, it is highly desirable to develop some rapid and effective computational methods to predict the structure of new proteins so as to expedite the process of deducing their function. All the structure prediction methods basically rely on the idea that there is a correlation between residue sequence and structure. The primary structure is unique for each protein and it is generally accepted that a protein’s primary structure is enough to determine its folding process to secondary, tertiary and quaternary structure. Despite recent efforts to develop automated protein structure determination protocols, structural genomic projects are slow in generating fold assignments for complete proteomes, and spatial structures remain unknown for many protein families. Alternative cheap and fast methods to assign folds using prediction algorithms continue to provide valuable structural information for many proteins. Protein structure determination and prediction has been a focal research subject in life sciences due to the importance of protein structure in understanding the biological and chemical activities of organisms/cell. This review comprehends the various recent advanced methods for protein structure predictions such as a two-stage method for assigning residues one of the three secondary structure states, prediction of homo-oligomeric proteins based on nearest neighbour algorithm, sequence–based hidden markov model, practical ab initio methods aimed at finding the native structure of the protein by simulating the biological process of protein folding, and metapredictors based on consensus form multiple methods.

Direct analysis of cooperativity in multisubunit allosteric proteins

Allosteric regulation of protein function is a fundamental phenomenon of major importance in many cellular processes. Such regulation is often achieved by ligand-induced conformational changes in multimeric proteins that may give rise to cooperativity in protein function. At the heart of allosteric mechanisms offered to account for such phenomenon, involving either concerted or sequential conformational transitions, lie changes in intersubunit interactions along the ligation pathway of the protein. However, structure-function analysis of such homooligomeric proteins by means of mutagenesis, although it provides valuable indirect information regarding (allosteric) mechanisms of action, it does not define the contribution of individual subunits nor interactions thereof to cooperativity in protein function, because any point mutation introduced into homooligomeric proteins will be present in all subunits. Here, we present a general strategy for the direct analysis of cooperativity in multisubunit proteins that combines measurement of the effects on protein function of all possible combinations of mutated subunits with analysis of the hierarchy of intersubunit interactions, assessed by using high-order double-mutant cycle-coupling analysis. We show that the pattern of high-order intersubunit coupling can serve as a discriminative criterion for defining concerted versus sequential conformational transitions underlying protein function. This strategy was applied to the particular case of the voltage-activated potassium channel protein (Kv) to provide compelling evidence for a concerted all-or-none activation gate opening of the Kv channel pore domain. An direct and detailed analysis of the contribution of high-order intersubunit interactions to cooperativity in the function of an allosteric protein has not previously been presented.

High-resolution structure determination of the CylR2 homodimer using paramagnetic relaxation enhancement and structure-based prediction of molecular alignment

Structure determination of homooligomeric proteins by NMR spectroscopy is difficult due to the lack of chemical shift perturbation data, which is very effective in restricting the binding interface in heterooligomeric systems, and the difficulty of obtaining a sufficient number of intermonomer distance restraints. Here we solved the high-resolution solution structure of the 15.4 kDa homodimer CylR2, the regulator of cytolysin production from Enterococcus faecalis, which deviates by 1.1 Å from the previously determined X-ray structure. We studied the influence of different experimental information such as long-range distances derived from paramagnetic relaxation enhancement, residual dipolar couplings, symmetry restraints and intermonomer Nuclear Overhauser Effect restraints on the accuracy of the derived structure. In addition, we show that it is useful to combine experimental information with methods of ab initio docking when the available experimental data are not sufficient to obtain convergence to the correct homodimeric structure. In particular, intermonomer distances may not be required when residual dipolar couplings are compared to values predicted on the basis of the charge distribution and the shape of ab initio docking solutions.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-007-9204-4) contains supplementary material, which is available to authorized users.

Antifungal activity of plant and bacterial ureases

Ureases (EC 3.5.1.5) are nickel-dependent metalloenzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. Produced by plants, fungi and bacteria, but not by animals, ureases share significant homology and similar mechanisms of catalysis, although differing in quaternary structures. While fungal and plant ureases are homo-oligomeric proteins of 90 kDa subunits, bacterial ureases are multimers of two (e.g. Helicobacter pylori) or three subunit complexes. It has been proposed that in plants these enzymes are involved in nitrogen bioavailability and in protection against pathogens. Previous studies by our group have shown that plant ureases, but not a bacterial ( Bacillus pasteurii) urease, display insecticidal activity. Herein we demonstrate that ( Glycine max) embryo-specific soybean urease, jackbean ( Canavalia ensiformis) major urease and a recombinant H. pylori urease impair growth of selected phytopathogenic fungi at sub-micromolar concentrations. This antifungal property of ureases is not affected by treatment of the proteins with an irreversible inhibitor of the ureolytic activity. Scanning electron microscopy of urease-treated fungi suggests plasmolysis and cell wall injuries. Altogether, our data indicate that ureases probably contribute to the plant arsenal of defense compounds against predators and phytopathogens and that the urease defense mechanism is independent of ammonia release from urea.

Prediction of homo-oligomeric proteins based on nearest neighbour algorithm

Nearest neighbour algorithm is applied to discriminating homo-oligomeric proteins from the protein primary structure. The algorithm describes protein primary sequences by their subsequence distributions, therefore, the information of protein sequences is sufficiently taken into account. When the length of subsequence increases, all performance measures have the tendency to ascend quickly for the tests of the method on the three data sets. Our tests demonstrate that the residue order along protein sequences plays an important role in recognition of the homo-oligomers, and nearest neighbour algorithm method is a simple and effective tool for classification of homo-oligomeric proteins. It is further confirmed that protein primary sequence encodes quaternary structure information.

Stepwise disassembly and apparent nonstepwise reassembly for the oligomeric RbsD protein

Many cellular proteins exist as homo-oligomers. The mechanism of the assembly process of such proteins is still poorly understood. We have previously observed that Hsp16.3, a protein exhibiting chaperone-like activity, undergoes stepwise disassembly and nonstepwise reassembly. Here, the disassembly and reassembly of a nonchaperone protein RbsD, from Escherichia coli, was studied in vitro. The protein was found to mainly exist as decamers with a small portion of apparently larger oligomeric forms, both of which are able to refold/reassemble effectively in a spontaneous way after being completely unfolded. Disassembly RbsD intermediates including pentamers, tetramers, trimers, dimers, and monomers were detected by using urea-containing pore gradient polyacrylamide gel electrophoresis, while only pentamers were detected for its reassembly. The observation of stepwise disassembly and apparent nonstepwise reassembly for both a chaperone protein (Hsp16.3) and a nonchaperone protein (RbsD) strongly suggests that such a feature is most likely general for homo-oligomeric proteins.