Mark E. Girvin

Structural changes linked to proton translocation by subunit c of the ATP synthase.

F1FO ATP synthases use a transmembrane proton gradient to drive the synthesis of cellular ATP. The structure of the cytosolic F1 portion of the enzyme and the basic mechanism of ATP hydrolysis by F1 are now well established, but how proton translocation through the transmembrane FO portion drives these catalytic changes is less clear. Here we describe the structural changes in the proton-translocating FO subunit c that are induced by deprotonating the specific aspartic acid involved in proton transport. Conformational changes between the protonated and deprotonated forms of subunit c provide the structural basis for an explicit mechanism to explain coupling of proton translocation by FO to the rotation of subunits within the core of F1. Rotation of these subunits within F1 causes the catalytic conformational changes in the active sites of F1 that result in ATP synthesis.

An approach to membrane protein structure without crystals

The lactose permease of Escherichia coli catalyzes coupled translocation of galactosides and H+ across the cell membrane. It is the best-characterized member of the Major Facilitator Superfamily, a related group of membrane proteins with 12 transmembrane domains that mediate transport of various substrates across cell membranes. Despite decades of effort and their functional importance in all kingdoms of life, no high-resolution structures have been solved for any member of this family. However, extensive biochemical, genetic, and biophysical studies on lactose permease have established its transmembrane topology, secondary structure, and numerous interhelical contacts. Here we demonstrate that this information is sufficient to calculate a structural model at the level of helix packing or better.

Mechanism of Constitutive Phosphoinositide 3-Kinase Activation by Oncogenic Mutants of the p85 Regulatory Subunit

p85/p110 phosphoinositide 3-kinases regulate multiple cell functions and are frequently mutated in human cancer. The p85 regulatory subunit stabilizes and inhibits the p110 catalytic subunit. The minimal fragment of p85 capable of regulating p110 is the N-terminal SH2 domain linked to the coiled-coil iSH2 domain (referred to as p85ni). We have previously proposed that the conformationally rigid iSH2 domain tethers p110 to p85, facilitating regulatory interactions between p110 and the p85 nSH2 domain. In an oncogenic mutant of murine p85, truncation at residue 571 leads to constitutively increased phosphoinositide 3-kinase activity, which has been proposed to result from either loss of an inhibitory Ser-608 autophosphorylation site or altered interactions with cellular regulatory factors. We have examined this mutant (referred to as p65) in vitro and find that p65 binds but does not inhibit p110, leading to constitutive p110 activity. This activated phenotype is observed with recombinant proteins in the absence of cellular factors. Importantly, this effect is also produced by truncating p85ni at residue 571. Thus, the phenotype is not because of loss of the Ser-608 inhibitory autophosphorylation site, which is not present in p85ni. To determine the structural basis for the phenotype of p65, we used a broadly applicable spin label/NMR approach to define the positioning of the nSH2 domain relative to the iSH2 domain. We found that one face of the nSH2 domain packs against the 581–593 region of the iSH2 domain. The loss of this interaction in the truncated p65 would remove the orienting constraints on the nSH2 domain, leading to a loss of p110 regulation by the nSH2. Based on these findings, we propose a general model for oncogenic mutants of p85 and p110 in which disruption of nSH2-p110 regulatory contacts leads to constitutive p110 activity.

Transition-state analogs as inhibitors of human and malarial hypoxanthine-guanine phosphoribosyltransferases

The proposed transition state for hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) has been used to design and synthesize powerful inhibitors that contain features of the transition state. The iminoribitols (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinHP) and (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinGP) are the most powerful inhibitors yet reported for both human and malarial HGPRTs. Equilibrium binding constants are >1,000-fold tighter than the binding of the nucleotide substrate. The NMR spectrum of malaria HGXPRT in the Michaelis complex reveals downfield hydrogen-bonded protons. The chemical shifts move farther downfield with bound inhibitor. The inhibitors are lead compounds for species-specific antibiotics against parasitic protozoa. The high-resolution crystal structure of human HGPRT with immucillinGP is reported in the companion paper.

MOLECULAR AND STRUCTURAL ANALYSIS OF A CONTINUOUS BIRCH PROFILIN EPITOPE DEFINED BY A MONOCLONAL ANTIBODY

The interaction of a mouse monoclonal antibody (4A6) and birch profilin, a structurally well conserved actin- and phosphoinositide-binding protein and cross-reactive allergen, was characterized. In contrast to serum IgE from allergic patients, which shows cross-reactivity with most plants, monoclonal antibody 4A6 selectively reacted with tree pollen profilins. Using synthetic overlapping peptides, a continuous hexapeptide epitope was identified. The exchange of a single amino acid (Gln-47 → Glu) within the epitope was found to abolish the binding of monoclonal antibody 4A6 to other plant profilins. The NMR analyses of the birch and the nonreactive timothy grass profilin peptides showed that the loss of binding was not due to major structural differences. Both peptides adopted extended conformations similar to that observed for the epitope in the x-ray crystal structure of the native birch profilin. Binding studies with peptides and birch profilin mutants generated by in vitro mutagenesis demonstrated that the change of Gln-47 to acidic amino acids (e.g. Glu or Asp) led to electrostatic repulsion of monoclonal antibody 4A6. In conclusion the molecular and structural analyses of the interaction of a monoclonal antibody with a continuous peptide epitope, recognized in a conformation similar to that displayed on the native protein, are presented.

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

We previously proposed a model of Class IA PI3K regulation in which p85 inhibition of p110α requires (i) an inhibitory contact between the p85 nSH2 domain and the p110α helical domain, and (ii) a contact between the p85 nSH2 and iSH2 domains that orients the nSH2 so as to inhibit p110α. We proposed that oncogenic truncations of p85 fail to inhibit p110 due to a loss of the iSH2-nSH2 contact. However, we now find that within the context of a minimal regulatory fragment of p85 (the nSH2-iSH2 fragment, termed p85ni), the nSH2 domain rotates much more freely (τc ≈12.7 ns) than it could if it were interacting rigidly with the iSH2 domain. These data are not compatible with our previous model. We therefore tested an alternative model in which oncogenic p85 truncations destabilize an interface between the p110α C2 domain (residue N345) and the p85 iSH2 domain (residues D560 and N564). p85ni-D560K/N564K shows reduced inhibition of p110α, similar to the truncated p85ni-572STOP. Conversely, wild-type p85ni poorly inhibits p110αN345K. Strikingly, the p110αN345K mutant is inhibited to the same extent by the wild-type or truncated p85ni, suggesting that mutation of p110α-N345 is not additive with the p85ni-572STOP mutation. Similarly, the D560K/N564K mutation is not additive with the p85ni-572STOP mutant for downstream signaling or cellular transformation. Thus, our data suggests that mutations at the C2-iSH2 domain contact and truncations of the iSH2 domain, which are found in human tumors, both act by disrupting the C2-iSH2 domain interface.

The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins

The New York Consortium on Membrane Protein Structure (NYCOMPS) was formed to accelerate the acquisition of structural information on membrane proteins by applying a structural genomics approach. NYCOMPS comprises a bioinformatics group, a centralized facility operating a high-throughput cloning and screening pipeline, a set of associated wet labs that perform high-level protein production and structure determination by x-ray crystallography and NMR, and a set of investigators focused on methods development. In the first three years of operation, the NYCOMPS pipeline has so far produced and screened 7,250 expression constructs for 8,045 target proteins. Approximately 600 of these verified targets were scaled up to levels required for structural studies, so far yielding 24 membrane protein crystals. Here we describe the overall structure of NYCOMPS and provide details on the high-throughput pipeline.

Letter to the Editor: Sequence-specific resonance assignment of the carboxyl terminal domain of Connexin43

The gap junction family of integral membrane proteins enables the direct cytoplasmic exchange of ions and small molecules (<1 kDa), including second messengers. Gap junctions are involved in a diverse array of cellular processes including cellular differentiation and development, metabolic homeostasis, and in excitable tissue, electrical coupling. They are formed by the apposition of connexons from adjacent cells, where each connexon is formed of six connexin proteins. Connexins are four transmembrane domain proteins with intracellular Nand C-terminal regions. More than twenty different mammalian connexins exist with the major divergence occurring in the cytoplasmic loop (CL) and carboxyl terminal (CT) domains. The subject of this paper, Cx43, is the most widely expressed gap junction protein and is essential for normal cardiac development and function. Recently, the idea of gap junctions being formed solely of the connexin proteins has been replaced by the concept that connexons may be centerpieces of a macromolecular complex or ‘Nexus’ (Spray et al., 1999). Integral tight junction, tight junctionassociated, cytoskeletal, adhedrens junctional complex and tyrosine kinase proteins bind to and/or modify the CT of Cx43, consistent with a more active role for gap junctions in cellular functions (Duffy et al., 2002a). A 7.5 A resolution structure of a recombinant Cx43 cardiac gap junction channel (Unger et al., 1999) has been solved; however, Cx43 was truncated to remove most of the CT. Such truncated constructs ∗To whom correspondence should be addressed. E-mail: girvin@aecom.yu.edu form functional channels, but pH sensitivity and interactions with the ‘Nexus’ proteins are altered. Separate co-expression of the CT domain partially restores pH sensitivity (Morley et al., 1996), and recombinant CT domain binds to the Nexus proteins (Duffy et al., 2002b). The hypothesis that acidification-induced uncoupling results from the intramolecular interaction between the CT domain and a separate region of the protein affiliated with the pore (Morley et al., 1996), was supported by our studies demonstrating pH dependent binding between the CT domain and a region of CL (L2) (Duffy et al., 2002c). Binding of L2 induced no significant chemical shift changes in 15N labeled CT, suggesting that the CT structure necessary for recognizing and binding CL is pre-formed. To understand the structural bases of connexin regulation, we are studying the Cx43CT in the two protonation states for which binding (pH 5.8) or no binding (pH 7.3) to L2 loop are observed. Here we report the sequence-specific assignments of the loop-binding Cx43CT conformation at pH 5.8. These assignments will be generally useful for mapping the binding sites for all of the Nexus proteins.

Structural Basis for the Physiological Temperature Dependence of the Association of VP16 with the Cytoplasmic Tail of Herpes Simplex Virus Glycoprotein H

ABSTRACT Critical events in the life cycle of herpes simplex virus (HSV) are the binding of cytoplasmic capsids to cellular organelles and subsequent envelopment. Work from several laboratories suggests that these events occur as a result of a network of partially redundant interactions among the capsid surface, tegument components, and cytoplasmic tails of virally encoded glycoproteins. Consistent with this model, we previously showed that tegument protein VP16 can specifically interact with the cytoplasmic tail of envelope protein gH in vitro and in vivo when fused to glutathione S-transferase and to green fluorescent protein, respectively. In both instances, this association was strikingly temperature dependent: binding occurred only at 37°C and not at lower temperatures. Here we demonstrate that virally expressed full-length gH and VP16 can be coimmunoprecipitated from HSV-infected cells and that this association is also critically dependent upon the physiological temperature. To investigate the basis of this temperature requirement, we performed one- and two-dimensional nuclear magnetic resonance spectroscopy on peptides with the sequence of the gH tail. We found that the gH tail is disorganized at temperatures permissive for binding but becomes structured at lower temperatures. Furthermore, a mutated tail unable to adopt this rigid conformation binds VP16 even at 4°C. We hypothesize that the gH tail is unstructured under physiological conditions in order to maximize the number of potential tegument partners with which it may associate. Being initially disordered, the gH tail may adopt one of several induced conformations as it associates with VP16 or alternative components of the tegument, maximizing redundancy during particle assembly.

Activated ERK2 Is a Monomer in Vitro with or without Divalent Cations and When Complexed to the Cytoplasmic Scaffold PEA-15

The extracellular signal-regulated protein kinase, ERK2, fully activated by phosphorylation and without a His(6) tag, shows little tendency to dimerize with or without either calcium or magnesium ions when analyzed by light scattering or analytical ultracentrifugation. Light scattering shows that ~90% of ERK2 is monomeric. Sedimentation equilibrium data (obtained at 4.8-11.2 μM ERK2) with or without magnesium (10 mM) are well described by an ideal one-component model with a fitted molar mass of 40180 ± 240 Da (without Mg(2+) ions) or 41290 ± 330 Da (with Mg(2+) ions). These values, close to the sequence-derived mass of 41711 Da, indicate that no significant dimerization of ERK2 occurs in solution. Analysis of sedimentation velocity data for a 15 μM solution of ERK2 with an enhanced van Holde-Weischet method determined the sedimentation coefficient (s) to be ~3.22 S for activated ERK2 with or without 10 mM MgCl(2). The frictional coefficient ratio (f/f(0)) of 1.28 calculated from the sedimentation velocity and equilibrium data is close to that expected for an ~42 kDa globular protein. The translational diffusion coefficient of ~8.3 × 10(-7) cm(2) s(-1) calculated from the experimentally determined molar mass and sedimentation coefficient agrees with the value determined by dynamic light scattering in the absence and presence of calcium or magnesium ions and a value determined by NMR spectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins [Casar, B., et al. (2008) Mol. Cell 31, 708-721]. Our light scattering data show, however, that ERK2 forms a strong 1:1 complex of ~57 kDa with the cytoplasmic scaffold protein PEA-15. Thus, ERK2 binds PEA-15 as a monomer. Our data provide strong evidence that ERK2 is monomeric under physiological conditions. Analysis of the same ERK2 construct with the nonphysiological His(6) tag shows substantial dimerization under the same ionic conditions.