Homo-oligomeric Protein Complexes Research Articles

DeepSub: Utilizing Deep Learning for Predicting the Number of Subunits in Homo-Oligomeric Protein Complexes.

The molecular weight (MW) of an enzyme is a critical parameter in enzyme-constrained models (ecModels). It is determined by two factors: the presence of subunits and the abundance of each subunit. Although the number of subunits (NS) can potentially be obtained from UniProt, this information is not readily available for most proteins. In this study, we addressed this gap by extracting and curating subunit information from the UniProt database to establish a robust benchmark dataset. Subsequently, we propose a novel model named DeepSub, which leverages the protein language model and Bi-directional Gated Recurrent Unit (GRU), to predict NS in homo-oligomers solely based on protein sequences. DeepSub demonstrates remarkable accuracy, achieving an accuracy rate as high as 0.967, surpassing the performance of QUEEN. To validate the effectiveness of DeepSub, we performed predictions for protein homo-oligomers that have been reported in the literature but are not documented in the UniProt database. Examples include homoserine dehydrogenase from Corynebacterium glutamicum, Matrilin-4 from Mus musculus and Homo sapiens, and the Multimerins protein family from M. musculus and H. sapiens. The predicted results align closely with the reported findings in the literature, underscoring the reliability and utility of DeepSub.

Spatial Structure of Lectin from the Mussel Mytilus trossulus: In-Sights from Molecular Modelling and Practical Proof.

Most proteins have the ability to self-associate into homooligomeric protein complexes, which consist of two or more identical subunits. Today, modern methods of molecular modeling are an integral part of the study of many biologically active molecules. In silico methods are widely used in structure establishing and function and activity prediction of lectins - carbohydrate-binding proteins. Here, we described by computer simulation the spatial organization of lectin isolated from the mantle of the mussel Mytilus trossulus (MTL). It was shown that the dimerization of MTL gives a total of six ligand binding sites that may be important for the manifestation its biological properties. The ability of MTL to form a dimeric and oligomeric structure was confirmed by dynamic light scattering and SDS-PAGE methods.

CoDNaS-Q: a database of conformational diversity of the native state of proteins with quaternary structure

A collection of conformers that exist in a dynamical equilibrium defines the native state of a protein. The structural differences between them describe their conformational diversity, a defining characteristic of the protein with an essential role in multiple cellular processes. Since most proteins carry out their functions by assembling into complexes, we have developed CoDNaS-Q, the first online resource to explore conformational diversity in homooligomeric proteins. It features a curated collection of redundant protein structures with known quaternary structure. CoDNaS-Q integrates relevant annotations that allow researchers to identify and explore the extent and possible reasons of conformational diversity in homooligomeric protein complexes. CoDNaS-Q is freely accessible at http://ufq.unq.edu.ar/codnasq/ or https://codnas-q.bioinformatica.org/home. The data can be retrieved from the website. The source code of the database can be downloaded from https://github.com/SfrRonaldo/codnas-q.

Accurate prediction of inter-protein residue-residue contacts for homo-oligomeric protein complexes.

Protein–protein interactions play a fundamental role in all cellular processes. Therefore, determining the structure of protein–protein complexes is crucial to understand their molecular mechanisms and develop drugs targeting the protein–protein interactions. Recently, deep learning has led to a breakthrough in intra-protein contact prediction, achieving an unusual high accuracy in recent Critical Assessment of protein Structure Prediction (CASP) structure prediction challenges. However, due to the limited number of known homologous protein–protein interactions and the challenge to generate joint multiple sequence alignments of two interacting proteins, the advances in inter-protein contact prediction remain limited. Here, we have proposed a deep learning model to predict inter-protein residue–residue contacts across homo-oligomeric protein interfaces, named as DeepHomo. Unlike previous deep learning approaches, we integrated intra-protein distance map and inter-protein docking pattern, in addition to evolutionary coupling, sequence conservation, and physico-chemical information of monomers. DeepHomo was extensively tested on both experimentally determined structures and realistic CASP-Critical Assessment of Predicted Interaction (CAPRI) targets. It was shown that DeepHomo achieved a high precision of >60% for the top predicted contact and outperformed state-of-the-art direct-coupling analysis and machine learning-based approaches. Integrating predicted inter-chain contacts into protein–protein docking significantly improved the docking accuracy on the benchmark dataset of realistic homo-dimeric targets from CASP-CAPRI experiments. DeepHomo is available at http://huanglab.phys.hust.edu.cn/DeepHomo/

Population Distributions from Native Mass Spectrometry Titrations Reveal Nearest-Neighbor Cooperativity in the Ring-Shaped Oligomeric Protein TRAP.

Allostery pervades macromolecular function and drives cooperative binding of ligands to macromolecules. To decipher the mechanisms of cooperative ligand binding, it is necessary to define, at a microscopic level, the thermodynamic consequences of binding of each ligand to its energetically coupled site(s). However, extracting these microscopic constants is difficult for macromolecules with more than two binding sites, because the observable [e.g., nuclear magnetic resonance (NMR) chemical shift changes, fluorescence, and enthalpy] can be altered by allostery, thereby distorting its proportionality to site occupancy. Native mass spectrometry (MS) can directly quantify the populations of homo-oligomeric protein species with different numbers of bound ligands, provided the populations are proportional to ion counts and that MS-compatible electrolytes do not alter the overall thermodynamics. These measurements can help decipher allosteric mechanisms by providing unparalleled access to the statistical thermodynamic partition function. We used native MS (nMS) to study the cooperative binding of tryptophan (Trp) to Bacillus stearothermophilus trp RNA binding attenuation protein (TRAP), a ring-shaped homo-oligomeric protein complex with 11 identical binding sites. MS-compatible solutions did not significantly perturb protein structure or thermodynamics as assessed by isothermal titration calorimetry and NMR spectroscopy. Populations of Trpn-TRAP11 states were quantified as a function of Trp concentration by nMS. The population distributions could not be explained by a noncooperative binding model but were described well by a mechanistic nearest-neighbor cooperative model. Nonlinear least-squares fitting yielded microscopic thermodynamic constants that define the interactions between neighboring binding sites. This approach may be applied to quantify thermodynamic cooperativity in other ring-shaped proteins.

Functional Implications of Multiple IM30 Oligomeric States.

The inner membrane-associated protein of 30 kDa (IM30), also known as the vesicle-inducing protein in plastids 1 (Vipp1), is essential for photo-autotrophic growth of cyanobacteria, algae and higher plants. While its exact function still remains largely elusive, it is commonly accepted that IM30 is crucially involved in thylakoid membrane biogenesis, stabilization and/or maintenance. A characteristic feature of IM30 is its intrinsic propensity to form large homo-oligomeric protein complexes. 15 years ago, it has been reported that these supercomplexes have a ring-shaped structure. However, the in vivo significance of these ring structures is not finally resolved yet and the formation of more complex assemblies has been reported. We here present and discuss research on IM30 conducted within the past 25 years with a special emphasis on the question of why we potentially need IM30 supercomplexes in vivo.

Proper evaluation of chemical cross-linking-based spatial restraints improves the precision of modeling homo-oligomeric protein complexes

BackgroundThe function of oligomeric proteins is inherently linked to their quaternary structure. In the absence of high-resolution data, low-resolution information in the form of spatial restraints can significantly contribute to the precision and accuracy of structural models obtained using computational approaches. To obtain such restraints, chemical cross-linking coupled with mass spectrometry (XL-MS) is commonly used. However, the use of XL-MS in the modeling of protein complexes comprised of identical subunits (homo-oligomers) is often hindered by the inherent ambiguity of intra- and inter-subunit connection assignment.ResultsWe present a comprehensive evaluation of (1) different methods for inter-residue distance calculations, and (2) different approaches for the scoring of spatial restraints. Our results show that using Solvent Accessible Surface distances (SASDs) instead of Euclidean distances (EUCs) greatly reduces the assignation ambiguity and delivers better modeling precision. Furthermore, ambiguous connections should be considered as inter-subunit only when the intra-subunit alternative exceeds the distance threshold. Modeling performance can also be improved if symmetry, characteristic for most homo-oligomers, is explicitly defined in the scoring function.ConclusionsOur findings provide guidelines for proper evaluation of chemical cross-linking-based spatial restraints in modeling homo-oligomeric protein complexes, which could facilitate structural characterization of this important group of proteins.

Systematic solution to homo-oligomeric structures determined by NMR.

Protein structure determination by NMR has predominantly relied on simulated annealing-based conformational search for a converged fold using primarily distance constraints, including constraints derived from nuclear Overhauser effects, paramagnetic relaxation enhancement, and cysteine crosslinkings. Although there is no guarantee that the converged fold represents the global minimum of the conformational space, it is generally accepted that good convergence is synonymous to the global minimum. Here, we show such a criterion breaks down in the presence of large numbers of ambiguous constraints from NMR experiments on homo-oligomeric protein complexes. A systematic evaluation of the conformational solutions that satisfy the NMR constraints of a trimeric membrane protein, DAGK, reveals 9 distinct folds, including the reported NMR and crystal structures. This result highlights the fundamental limitation of global fold determination for homo-oligomeric proteins using ambiguous distance constraints and provides a systematic solution for exhaustive enumeration of all satisfying solutions.

Solution and Crystallographic Structures of the Central Region of the Phosphoprotein from Human Metapneumovirus

Human metapneumovirus (HMPV) of the family Paramyxoviridae is a major cause of respiratory illness worldwide. Phosphoproteins (P) from Paramyxoviridae are essential co-factors of the viral RNA polymerase that form tetramers and possess long intrinsically disordered regions (IDRs). We located the central region of HMPV P (Pced) which is involved in tetramerization using disorder analysis and modeled its 3D structure ab initio using Rosetta fold-and-dock. We characterized the solution-structure of Pced using small angle X-ray scattering (SAXS) and carried out direct fitting to the scattering data to filter out incorrect models. Molecular dynamics simulations (MDS) and ensemble optimization were employed to select correct models and capture the dynamic character of Pced. Our analysis revealed that oligomerization involves a compact central core located between residues 169-194 (Pcore), that is surrounded by flexible regions with α-helical propensity. We crystallized this fragment and solved its structure at 3.1 Å resolution by molecular replacement, using the folded core from our SAXS-validated ab initio model. The RMSD between modeled and experimental tetramers is as low as 0.9 Å, demonstrating the accuracy of the approach. A comparison of the structure of HMPV P to existing mononegavirales Pced structures suggests that Pced evolved under weak selective pressure. Finally, we discuss the advantages of using SAXS in combination with ab initio modeling and MDS to solve the structure of small, homo-oligomeric protein complexes.

A graphical method for analyzing distance restraints using residual dipolar couplings for structure determination of symmetric protein homo‐oligomers

High-resolution structure determination of homo-oligomeric protein complexes remains a daunting task for NMR spectroscopists. Although isotope-filtered experiments allow separation of intermolecular NOEs from intramolecular NOEs and determination of the structure of each subunit within the oligomeric state, degenerate chemical shifts of equivalent nuclei from different subunits make it difficult to assign intermolecular NOEs to nuclei from specific pairs of subunits with certainty, hindering structural analysis of the oligomeric state. Here, we introduce a graphical method, DISCO, for the analysis of intermolecular distance restraints and structure determination of symmetric homo-oligomers using residual dipolar couplings. Based on knowledge that the symmetry axis of an oligomeric complex must be parallel to an eigenvector of the alignment tensor of residual dipolar couplings, we can represent distance restraints as annuli in a plane encoding the parameters of the symmetry axis. Oligomeric protein structures with the best restraint satisfaction correspond to regions of this plane with the greatest number of overlapping annuli. This graphical analysis yields a technique to characterize the complete set of oligomeric structures satisfying the distance restraints and to quantitatively evaluate the contribution of each distance restraint. We demonstrate our method for the trimeric E. coli diacylglycerol kinase, addressing the challenges in obtaining subunit assignments for distance restraints. We also demonstrate our method on a dimeric mutant of the immunoglobulin-binding domain B1 of streptococcal protein G to show the resilience of our method to ambiguous atom assignments. In both studies, DISCO computed oligomer structures with high accuracy despite using ambiguously assigned distance restraints.

Pun1p is a metal ion-inducible, calcineurin/Crz1p-regulated plasma membrane protein required for cell wall integrity

Under conditions of environmental stress, the plasma membrane is involved in several regulatory processes to promote cell survival, like maintenance of signaling pathways, cell wall organization and intracellular ion homeostasis. PUN1 encodes a plasma membrane protein localizing to the ergosterol-rich membrane compartment occupied also by the arginine permease Can1. We found that the PUN1 (YLR414c) gene is transcriptionally induced upon metal ion stress. Northern blot analysis of the transcriptional regulation of PUN1 showed that the calcium dependent transcription factor Crz1p is required for PUN1 induction upon heavy metal stress. Here we report that mutants deleted for PUN1 exhibit increased metal ion sensitivity and morphological abnormalities. Microscopical and ultrastructural observations revealed a severe cell wall defect of pun1∆ mutants. By using chemical cross-linking, Blue native electrophoresis, and co-immunoprecipitation we found that Pun1p forms homo-oligomeric protein complexes. We propose that Pun1p is a stress-regulated factor required for cell wall integrity, thereby expanding the functional significance of lateral plasma membrane compartments.

Building Macromolecular Assemblies by Information-driven Docking

Over the last years, large scale proteomics studies have generated a wealth of information of biomolecular complexes. Adding the structural dimension to the resulting interactomes represents a major challenge that classical structural experimental methods alone will have difficulties to confront. To meet this challenge, complementary modeling techniques such as docking are thus needed. Among the current docking methods, HADDOCK (High Ambiguity-Driven DOCKing) distinguishes itself from others by the use of experimental and/or bioinformatics data to drive the modeling process and has shown a strong performance in the critical assessment of prediction of interactions (CAPRI), a blind experiment for the prediction of interactions. Although most docking programs are limited to binary complexes, HADDOCK can deal with multiple molecules (up to six), a capability that will be required to build large macromolecular assemblies. We present here a novel web interface of HADDOCK that allows the user to dock up to six biomolecules simultaneously. This interface allows the inclusion of a large variety of both experimental and/or bioinformatics data and supports several types of cyclic and dihedral symmetries in the docking of multibody assemblies. The server was tested on a benchmark of six cases, containing five symmetric homo-oligomeric protein complexes and one symmetric protein-DNA complex. Our results reveal that, in the presence of either bioinformatics and/or experimental data, HADDOCK shows an excellent performance: in all cases, HADDOCK was able to generate good to high quality solutions and ranked them at the top, demonstrating its ability to model symmetric multicomponent assemblies. Docking methods can thus play an important role in adding the structural dimension to interactomes. However, although the current docking methodologies were successful for a vast range of cases, considering the variety and complexity of macromolecular assemblies, inclusion of some kind of experimental information (e.g. from mass spectrometry, nuclear magnetic resonance, cryoelectron microscopy, etc.) will remain highly desirable to obtain reliable results.

Homomeric protein complexes: evolution and assembly

Homo-oligomeric protein complexes are functionally vital and highly abundant in living cells. In the present article, we review our current understanding of their geometry and evolution, including aspects of the symmetry of these complexes and their interaction interfaces. Also, we briefly discuss the pathway of their assembly in solution.

Emergence of symmetry in homooligomeric biological assemblies

Naturally occurring homooligomeric protein complexes exhibit striking internal symmetry. The evolutionary origins of this symmetry have been the subject of considerable speculation; proposals for the advantages associated with symmetry include greater folding efficiency, reduced aggregation, amenability to allosteric regulation, and greater adaptability. An alternative possibility stems from the idea that to contribute to fitness, and hence be subject to evolutionary optimization, a complex must be significantly populated, which implies that the interaction energy between monomers in the ancestors of modern-day complexes must have been sufficient to at least partially overcome the entropic cost of association. Here, we investigate the effects of this bias toward very-low-energy complexes on the distribution of symmetry in primordial homooligomers modeled as randomly interacting pairs of monomers. We demonstrate quantitatively that a bias toward very-low-energy complexes can result in the emergence of symmetry from random ensembles in which the overall frequency of symmetric complexes is vanishingly small. This result is corroborated by using explicit protein-protein docking calculations to generate ensembles of randomly docked complexes: the fraction of these that are symmetric increases from 0.02% in the overall population to >50% in very low energy subpopulations.

Efficiency of Resonance Energy Transfer in Homo-Oligomeric Complexes of Proteins

A theoretical model is proposed for the apparent efficiency of fluorescence (Förster) resonance energy transfer (FRET) in mixtures of free monomers and homo-oligomeric protein complexes of uniform size. The model takes into account possible pathways for transfer of optical excitations from single donors to multiple acceptors and from multiple donors (non-simultaneously) to single acceptors. This necessary departure from the standard theory has been suggested in the literature, but it has only been successfully implemented for a few particular cases, such as for particular geometries of the oligomers. The predictions of the present theoretical model differ significantly from those of the standard theory, with the exception of the case of dimers, for which agreement is observed. This model therefore provides new insights into the FRET behavior of oligomers comprising more than two monomers, and also suggests means for determining the size of oligomeric protein complexes as well as the proportion of associated and unassociated monomers.

“Zero‐length” cross‐linking in solid state as an approach for analysis of protein–protein interactions

We have developed a new approach for the analysis of interacting interfaces in protein complexes and protein quaternary structure based on cross-linking in the solid state. Protein complexes are freeze-dried under vacuum, and cross-links are introduced in the solid phase by dehydrating the protein in a nonaqueous solvent creating peptide bonds between amino and carboxyl groups of the interacting peptides. Cross-linked proteins are digested into peptides with trypsin in both H2(16)O and H(2)18O and then readily distinguished in mass spectra by characteristic 8 atomic mass unit (amu) shifts reflecting incorporation of two 18O atoms into each C terminus of proteolytic peptides. Computer analysis of mass spectrometry (MS) and MS/MS data is used to identify the cross-linked peptides. We demonstrated specificity and reproducibility of our method by cross-linking homo-oligomeric protein complexes of glutathione-S-transferase (GST) from Schistosoma japonicum alone or in a mixture of many other proteins. Identified cross-links were predominantly of amide origin, but six esters and thioesters were also found. The cross-linked peptides were validated against the GST monomer and dimer X-ray structures and by experimental (MS/MS) analyses. Some of the identified cross-links matched interacting peptides in the native 3D structure of GST, indicating that the structure of GST and its oligomeric complex remained primarily intact after freeze-drying. The pattern of oligomeric GST obtained in solid state was the same as that obtained in solution by Ru (II) Bpy(3)2+ catalyzed, oxidative "zero-length" cross-linking, confirming that it is feasible to use our strategy for analyzing the molecular interfaces of interacting proteins or peptides.

RS1, a Discoidin Domain-containing Retinal Cell Adhesion Protein Associated with X-linked Retinoschisis, Exists as a Novel Disulfide-linked Octamer

RS1, also known as retinoschisin, is an extracellular protein that plays a crucial role in the cellular organization of the retina. Mutations in RS1 are responsible for X-linked retinoschisis, a common, early-onset macular degeneration in males that results in a splitting of the inner layers of the retina and severe loss in vision. RS1 is assembled and secreted from photoreceptors and bipolar cells as a homo-oligomeric protein complex. Each subunit consists of a 157-amino acid discoidin domain flanked by two small segments of 39 and 5 amino acids. To begin to understand how the structure of RS1 relates to its role in retinal cell adhesion and X-linked retinoschisis, we have determined the subunit organization and disulfide bonding pattern of RS1 by SDS gel electrophoresis, velocity sedimentation, and mass spectrometry. Our results indicate that RS1 exists as a novel octamer in which the eight subunits are joined together by Cys(59)-Cys(223) intermolecular disulfide bonds. Subunits within the octamer are further organized into dimers mediated by Cys(40)-Cys(40) bonds. These cysteines lie just outside the discoidin domain indicating that these flanking segments primarily function in the octamerization of RS1. Within the discoidin domain, two cysteine pairs (Cys(63)-Cys(219) and Cys(110)-Cys(142)) form intramolecular disulfide bonds that are important in protein folding, and one cysteine (Cys(83)) exists in its reduced state. Because mutations that disrupt subunit assembly cause X-linked retinoschisis, the assembly of RS1 into a disulfide-linked homo-octamer appears to be critical for its function as a retinal cell adhesion protein.

Ubiquitous soluble Mg 2+-ATPase complex : A structural study

We have performed a detailed structural analysis of the soluble Mg 2+-ATPase complex purified from Xenopus laevis ovary, which is an abundant and ubiquitous homo-oligomeric protein complex located in the nucleus and in the cytoplasm, belonging to a novel multigene-family of putative Mg 2+-ATPases. Enzyme activity staining after non-denaturing polyacrylamide gel electrophoresis revealed that Mg 2+-ATPase activity of the native protein is dependent on oligomerization and could not be detected in dissociated subunits. For the native protein a sedimentation coefficient of 15·3 S and a corresponding relative molecular mass of 612,000 was determined by analytical ultracentrifugation and a relative molecular mass of 590,000 was estimated from scanning transmission electron microscopy, supporting our previous conclusion that the oligomer comprises six 97,000 M r subunits. Conventional electron microscopy of negatively stained specimens revealed the Mg 2+-ATPase complex to be a hexagonal molecule in its favoured “end-on” projection and a double-banded molecule in its “side-on” projection (approx. 12 nm diameter; approx. 9 nm height). In addition, dimerized complexes could be observed in negatively stained specimens, yielding pronounced hexameric images and four-banded images in their end-on and side-on orientations, respectively (approx. 12 nm diameter; approx. 18·5 nm height). Two-dimensional (2D = mono-molecular) crystals have been produced from the dimerized complexes by the negative staining carbon film technique. Hexagonal crystals with a p6 plane group symmetry were obtained from molecules in their end-on orientation and longitudinal arrays with a p2 symmetry from complexes in their side-on orientation. A low-resolution molecular model of the native protein, derived from averages of these two 2D crystals, is presented. From our results we propose oligomerization as an inherent structural principle of organization for this whole newly defined Mg 2+-ATPase multigene-family, that includes such seemingly diverse functionally defined proteins as mammalian and yeast “vesicle fusion” and “peroxisome assembly” proteins and the product of the yeast cell cycle gene CDC48.

Two-dimensional crystallization, transmission electron microscopy and image processing of keyhole Limpet Haemocyanin (KLH)

KLH, the respiratory protein haemocyanin of the keyhole limpet Megathura crenulata, has been obtained as a high g pellet from the cell-free haemolymph and purified by gel permeation chromatography on a Biogel A15m column. The leading major protein peak eluted has been found to contain haemocyanin multi-decamers, followed by a second major peak containing single di-decamers, with small amounts of decamer and partly dissociated material following in the later fractions. The purified KLH di-decamer has been for two- dimensional crystallization studies with the negative staining-carbon film technique, in the presence of polyethylene glycol. In the side-on orientation, KLH has been found to produce two-dimensional crystals with a half-molecule linear displacement in consecutive rows. This is shown to be due to a specific association and two-dimensional crystal nucleation of the molecules in this arrangement. When oriented end-on, KLH has been found to form close- packed hexagonal monomolecular arrays which are not truly crystalline. This is because the five-fold rotational symmetry of the cylindrical macromolecule is not readily compatible with the hexagonal molecular packing. Computer-processed averaged images have been produced from the side-on KLH two-dimensional crystals and the end-on arrays, which reveal the principal molecular features of this homo-oligomeric protein complex to a resolution of ca 2.7 nm.