Transient Homodimers Research Articles

On the abundance and importance of AXXXA sequence motifs in globular proteins and their involvement in Cβ[sbnd]Cβ interaction

The AXXXA and GXXXG motifs are frequently observed in helices, especially in membrane proteins. The motif GXXXG is known to stabilize helix-helix association in membrane proteins via CαHO bonding. AXXXA sequence motif additionally stabilizes the folded state of proteins. We found 27,000 and 18,000 occurrences of AXXXA and GXXXG motifs in a non-redundant set of 6000 obligate homodimeric (OD) complexes. Interestingly, this is less pronounced in transient homodimers (TD) and heterodimers (HetD). On average each obligate homodimer contains four AXXXA motifs, it is 2 and 3.5 for HetD and TD, respectively. Focusing on the binding surface it is seen that 27 % of the ODs contain at least one AXXXA motif at the interface, whereas it is 17 % and 15 % for HetD and TD respectively. AXXXA predominantly stabilizes the OD quaternary structure via the side chain CβCβ interactions. This interaction is energetically favorable and is found to be a major driving force for OD quaternary structure stability. Cβ-Cβ interactions are observed ∼6 times higher than the known CαHO interaction for helix-helix stabilization. Two additional new interactions of CβO and OO are observed at the AXXXA containing interface regions. The occurrence of the motif gets drastically reduced if any of the terminal Ala residues are replaced by Gly. Our findings show the importance of AXXXA in providing stability to the quaternary structure through specific hydrophobic interactions and the specificity of the Ala residue at motif termini. The knowledge gained can be used for designing synthetic proteins of improved stability and for designing peptide-based therapeutics.

An atypical BRCT-BRCT interaction with the XRCC1 scaffold protein compacts human DNA Ligase IIIα within a flexible DNA repair complex.

The XRCC1–DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT–BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS–MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT–BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.

Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists

Although class A G protein-coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer-dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.

New insights into the organization of plasma membrane and its role in signaltransduction.

Plasma membranes have heterogeneous structures for efficient signal transduction, required to perform cell functions. Recent evidence indicates that the heterogeneous structures are produced by (1) compartmentalization by actin-based membrane skeleton, (2) raft domains, (3) receptor-receptor interactions, and (4) the binding of receptors to cytoskeletal proteins. This chapter provides an overview of recent studies on diffusion, clustering, raft association, actin binding, and signal transduction of membrane receptors, especially glycosylphosphatidylinositol (GPI)-anchored receptors. Studies on diffusion of GPI-anchored receptors suggest that rafts may be small and/or short-lived in plasma membranes. In steady state conditions, GPI-anchored receptors form transient homodimers, which may represent the "standby state" for the stable homodimers and oligomers upon ligation. Furthermore, It is proposed that upon ligation, the binding of GPI-anchored receptor clusters to cytoskeletal actin filaments produces a platform for downstream signaling, and that the pulse-like signaling easily maintains the stability of the overall signaling activity.

A functional feature analysis on diverse protein-protein interactions: application for the prediction of binding affinity.

Protein-protein interactions (PPIs) play crucial roles in diverse cellular processes. There are different types of PPIs based on the composition, affinity and whether the association is permanent or transient. Analyzing the diversity of PPIs at the atomic level is crucial for uncovering the key features governing the interactions involved in PPI. A systematic physico-chemical and conformational studies were implemented on interfaces involved in different PPIs, including crystal packing, weak transient heterodimers, weak transient homodimers, strong transient heterodimers and homodimers. The comparative analysis shows that the interfaces tend to be larger, less planar, and more tightly packed with the increase of the interaction strength. Meanwhile the strong interactions undergo greater conformational changes than the weak ones involving main chains as well as side chains. Finally, using 18 features derived from our analysis, we developed a support vector regression model to predict the binding affinity with a promising result, which further demonstrate the reliability of our studies. We believe this study will provide great help in more thorough understanding the mechanism of diverse PPIs.

Transient GPI-anchored protein homodimers are units for raft organization and function

Advanced single-molecule fluorescent imaging was applied to study the dynamic organization of raft-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the plasma membrane and their stimulation-induced changes. In resting cells, virtually all of the GPI-APs are mobile and continually form transient (~200 ms) homodimers (termed homodimer rafts) through ectodomain protein interactions, stabilized by the presence of the GPI-anchoring chain and cholesterol. Heterodimers do not form, suggesting a fundamental role for the specific ectodomain protein interaction. Under higher physiological expression conditions , homodimers coalesce to form hetero- and homo-GPI-AP oligomer rafts through raft-based lipid interactions. When CD59 was ligated, it formed stable oligomer rafts containing up to four CD59 molecules, which triggered intracellular Ca(2+) responses that were dependent on GPI anchorage and cholesterol, suggesting a key part played by transient homodimer rafts. Transient homodimer rafts are most likely one of the basic units for the organization and function of raft domains containing GPI-APs.

Transient Homodimer Interactions Studied Using the Electron Self-exchange Reaction

Transient homodimer protein interactions have been investigated by analyzing the influence of ionic strength (NaCl) on the electron self-exchange (the bimolecular reaction whereby the two oxidation states of a redox protein interconvert) rate constant (k(ese)) of four plastocyanins. The k(ese) values for the plastocyanins from spinach, Dryopteris crassirhizoma (a fern), and the green alga Ulva pertusa, which possess acidic patches of varying size and locations, increase 190-, 29-, and 21-fold, respectively, at elevated ionic strength (I = 2.03 M). In contrast, the k(ese) for the almost neutral cyanobacterial plastocyanin from Anabaena variabilis exhibits very little dependence on ionic strength. The temperature dependence of the k(ese) for spinach plastocyanin (I = 0.28 M) provides evidence for poor packing at the homodimer interface. Representative structures of the transient homodimers involved in electron self-exchange, which are consistent with fits of the ionic strength dependence of k(ese) to van Leeuwen theory, have been obtained from protein modeling and docking simulations. The Coulombic energy of the docked homodimers follows the order spinach > D. crassirhizoma > U. pertusa > A. variabilis, which matches that of the overall influence of ionic strength on k(ese). Analysis of the homodimer structures indicates that poor packing and high planarity are features of the interface that favor transient interactions. The physiologically relevant Mg2+ ion has a much more pronounced influence on the k(ese) of spinach plastocyanin, which along with the known properties of the thylakoid lumen suggests a biological role for electron self-exchange.

Structural Characterisation and Functional Significance of Transient Protein–Protein Interactions

Protein–protein complexes that dissociate and associate readily, often depending on the physiological condition or environment, play an important role in many biological processes. In order to characterise these ”transient” protein–protein interactions, two sets of complexes were collected and analysed. The first set consists of 16 experimentally validated “weak” transient homodimers, which are known to exist as monomers and dimers at physiological concentration, with dissociation constants in the micromolar range. A set of 23 functionally validated transient (i.e. intracellular signalling) heterodimers comprise the second set. This set includes complexes that are more stable, with nanomolar binding affinities, and require a molecular trigger to form and break the interaction. In comparison to more stable homodimeric complexes, the weak homodimers demonstrate smaller contact areas between protomers and the interfaces are more planar and polar on average. The physicochemical and geometrical properties of these weak homodimers more closely resemble those of non-obligate hetero-oligomeric complexes, whose components can exist either as monomers or as complexes in vivo. In contrast to the weak transient dimers, “strong” transient dimers often undergo large conformational changes upon association/dissociation and are characterised with larger, less planar and sometimes more hydrophobic interfaces. From sequence alignments we find that the interface residues of the weak transient homodimers are generally more conserved than surface residues, consistent with being constrained to maintain the protein–protein interaction during evolution. Protein families that include members with different oligomeric states or structures are identified, and found to exhibit a lower sequence conservation at the interface. The results are discussed in terms of the physiological function and evolution of protein–protein interactions.