Regulation Of Protein-protein Interactions Research Articles

The Crystal Structure of the α-Neurexin-1 Extracellular Region Reveals a Hinge Point for Mediating Synaptic Adhesion and Function

α- and β-neurexins (NRXNs) are transmembrane cell adhesion proteins that localize to presynaptic membranes in neurons and interact with the postsynaptic neuroligins (NLGNs). Their gene mutations are associated with the autism spectrum disorders. The extracellular region of α-NRXNs, containing nine independently folded domains, has structural complexity and unique functional characteristics, distinguishing it from the smaller β-NRXNs. We have solved the X-ray crystal structure of seven contiguous domains of the α-NRXN-1 extracellular region at 3.0 Å resolution. The structure reveals an arrangement where the N-terminal five domains adopt a more rigid linear conformation and the two C-terminal domains form a separate arm connected by a flexible hinge. In an extended conformation the molecule is suitably configured to accommodate a bound NLGN molecule, as supported by structural comparison and surface plasmon resonance. These studies provide the structural basis for a multifunctional synaptic adhesion complex mediated by α-NRXN-1.

Regulation of Protein-protein Interaction via Assembly of Coiled-coil Domain

The protein-protein interaction presides the various biological events in life. Toward the understanding of their functions and networks, various techniques to regulate the specific protein functions are developed and applied so far. Here we examined the novel method to regulate the protein-protein interactions via coiled-coil assembly.

Discovery of Selective Small-Molecule CD80 Inhibitors

Protein-protein interactions are widely found in biological systems controlling diverse cellular events. Because these interactions are implicated in many diseases such as autoimmunity and cancer, regulation of protein-protein interactions provides ideal targets for drug intervention. The CD80-CD28 costimulatory pathway plays a critical role in regulation of the immune response and thus constitutes an attractive target for therapeutic manipulation of autoimmune diseases. The objective of this study is to identify small compounds disrupting these pivotal protein-protein interactions. Compounds that specifically blocked binding of CD80 to CD28 were identified using a strategy involving a cell-based scintillation proximity assay as the initial step. Secondary screening (e.g., by analyzing the direct binding of these compounds to the target immobilized on a biosensor surface) revealed that these compounds are highly selective CD80 binders. Screening of structurally related derivatives led to the identification of the chemical features required for inhibition of the CD80-CD28 interaction. In addition, the optimization process led to a 10-fold increase in binding affinity of the CD80 inhibitors. Using this approach, the authors identify low-molecular-weight compounds that specifically and with high potency inhibit the interaction between CD80 and CD28. These compounds serve as promising starting points for further development of CD80 inhibitors as potential immunomodulatory drugs.

Pias1 Interaction and Sumoylation of Metabotropic Glutamate Receptor 8

Group III presynaptic metabotropic glutamate receptors (mGluRs) play a central role in regulating presynaptic activity through G-protein effects on ion channels and signal transducing enzymes. Like all Class C G-protein-coupled receptors, mGluR8 has an extended intracellular C-terminal domain (CTD) presumed to allow for modulation of downstream signaling. In a yeast two-hybrid screen of an adult rat brain cDNA library with the CTDs of mGluR8a and 8b (mGluR8-C) as baits, we identified sumo1 and four different components of the sumoylation cascade (ube2a, Pias1, Piasgamma, Piasxbeta) as interacting proteins. Binding assays using recombinant GST fusion proteins confirmed that Pias1 interacts not only with mGluR8-C but also with all group III mGluR CTDs. Pias1 binding to mGluR8-C required a region N-terminal to a consensus sumoylation motif and was not affected by arginine substitution of the conserved lysine 882 within this motif. Co-transfection of fluorescently tagged mGluR8a-C, sumo1, and enzymes of the sumoylation cascade into HEK293 cells showed that mGluR8a-C can be sumoylated in vivo. Arginine substitution of lysine 882 within the consensus sumoylation motif, but not other conserved lysines within the CTD, abolished in vivo sumoylation. Our results are consistent with post-translational sumoylation providing a novel mechanism of group III mGluR regulation.

Electrostatic properties of the structure of the docking and dimerization domain of protein kinase A IIalpha.

The structure of the N-terminal docking and dimerization domain of the type IIalpha regulatory subunit (RIIalpha D/D) of protein kinase A (PKA) forms a noncovalent stand-alone X-type four-helix bundle structural motif, consisting of two helix-loop-helix monomers. RIIalpha D/D possesses a strong hydrophobic core and two distinct, exposed faces. A hydrophobic face with a groove is the site of protein-protein interactions necessary for subcellular localization. A highly charged face, opposite to the former, may be involved in regulation of protein-protein interactions as a result of changes in phosphorylation state of the regulatory subunit. Although recent studies have addressed the hydrophobic character of packing of RIIalpha D/D and revealed the function of the hydrophobic face as the binding site to A-kinase anchoring proteins (AKAPs), little attention has been paid to the charges involved in structure and function. To examine the electrostatic character of the structure of RIIalpha D/D we have predicted mean apparent pKa values, based on Poisson-Boltzmann electrostatic calculations, using an ensemble of calculated dimer structures. We propose that the helix promoting sequence Glu34-X-X-X-Arg38 stabilizes the second helix of each monomer, through the formation of a (i, i +4) side chain salt bridge. We show that a weak inter-helical hydrogen bond between Tyr35-Glu19 of each monomer contributes to tertiary packing and may be responsible for discriminating from alternative quaternary packing of the two monomers. We also show that an inter-monomer hydrogen bond between Asp30-Arg40 contributes to quaternary packing. We propose that the charged face comprising of Asp27-Asp30-Glu34-Arg38-Arg40-Glu41-Arg43-Arg44 may be necessary to provide flexibility or stability in the region between the C-terminus and the interdomain/autoinhibitory sequence of RIIalpha, depending on the activation state of PKA. We also discuss the structural requirements necessary for the formation of a stacked (rather than intertwined) dimer, which has consequences for the orientation of the functionally important and distinct faces.

SUMO--nonclassical ubiquitin.

SUMO (small ubiquitin-related modifier) is the best-characterized member of a growing family of ubiquitin-related proteins. It resembles ubiquitin in its structure, its ability to be ligated to other proteins, as well as in the mechanism of ligation. However, in contrast to ubiquitination-often the first step on a one-way road to protein degradation-SUMOlation does not seem to mark proteins for degradation. In fact, SUMO may even function as an antagonist of ubiquitin in the degradation of selected proteins. While most SUMO targets are still at large, available data provide compelling evidence for a role of SUMO in the regulation of protein-protein interactions and/or subcellular localization.

SH2 and SH3 domains: From structure to function

Tony Pawson and Gerald D. Gish Division of Molecular and Developmental Biology Samuel Lunenfeld Research Institute Mt. Sinai Hospital Toronto, Ontario M5G 1X5 Canada Growth factors that function by activating receptors with protein-tyrosine kinase activity can elicit a wide range of biochemical responses, leading ultimately to changes in the proliferation, architecture, differentiation, and metabo- lism of their target cells. The means by which receptor tyrosine kinases select their targets and thereby stimulate specific intracellular signaling pathways has been illumi- nated by the identification of a conserved protein module of approximately 100 amino acids, the Src homology 2 (SH2) domain, which is found in a remarkably diverse group of cytoplasmic signaling proteins (Figure 1) and plays a pivotal role in their interactions with receptor tyro- sine kinases (Koch et al., 1991). Proteins with SH2 do- mains frequently possess a distinct sequence of about 50 residues, the SH3 domain, which is also implicated in the regulation of protein-protein interactions during signal transduction (see Clark et al., 1992, and references therein). Upon binding of an external ligand, growth factor recep- tors undergo dimerization, followed by the autophosphory- lation of specific tyrosine residues. Receptor autophos- phorylation acts as a molecular switch to create binding sites for the SH2 domains of cytoplasmic signaling pro- teins (Anderson et al., 1990), which thereby become tar- gets for activation. SH2 domains directly recognize phos- photyrosine (Matsuda et al., 1990). However, high affinity binding of an SH2 domain requires that the phosphotyro- sine be embedded within a specific amino acid sequence, as originally suggested by an examination of SH2-binding sites (Cantley et al., 1991). For example, the SH2-con- taining proteins phosphatidyfinositol (PI) 3’-kinase, the Ras GTP-ase-activating protein, and phospholipase Cy each bind to different autophosphorylated sites on the f3 recep- tor for platelet-derived growth factor (Kashishian et al., 1992; Fantl et al., 1992). Inhibition studies using small phosphotyrosine-contain- ing peptides have shown that molecules containing only 5 residues can effectively block SHBmediated binding to activated receptors (Fantl et al., 1992). The residues im- mediately C-terminal to the phosphotyrosine, especially those at the +l and +3 positions, appear to provide selec- tivity for specific SH2 domains. The dissociation constants for the interactions of SH2 domains with activated recep- tors or phosphotyrosine-containing peptides of optimal se- quence are generally estimated to be in the low nanomolar range, whereas binding to phosphotyrosine alone is much weaker (e.g., Mayer et al., 1992). In principle then, the ability of a given receptor to bind specific SHP-containing proteins (and thereby activate the corresponding signaling