Stephen R. Sprang

The structure of the G protein heterotrimer Giα1β1γ2

The crystallographic structure of the G protein heterotrimer Gi alpha 1(GDP)beta 1 gamma 2 (at 2.3 A) reveals two nonoverlapping regions of contact between alpha and beta, an extended interface between beta and nearly all of gamma, and limited interaction of alpha with gamma. The major alpha/beta interface covers switch II of alpha, and GTP-induced rearrangement of switch II causes subunit dissociation during signaling. Alterations in GDP binding in the heterotrimer (compared with alpha-GDP) explain stabilization of the inactive conformation of alpha by beta gamma. Repeated WD motifs in beta form a circularized sevenfold beta propeller. The conserved cores of these motifs are a scaffold for display of their more variable linkers on the exterior face of each propeller blade.

Structure of RGS4 Bound to AlF4−-Activated Giα1: Stabilization of the Transition State for GTP Hydrolysis

RGS proteins are GTPase activators for heterotrimeric G proteins. We report here the 2.8 A resolution crystal structure of the RGS protein RGS4 complexed with G(i alpha1)-Mg2+-GDP-AlF4 . Only the core domain of RGS4 is visible in the crystal. The core domain binds to the three switch regions of G(i alpha1), but does not contribute catalytic residues that directly interact with either GDP or AlF4-. Therefore, RGS4 appears to catalyze rapid hydrolysis of GTP primarily by stabilizing the switch regions of G(i alpha1), although the conserved Asn-128 from RGS4 could also play a catalytic role by interacting with the hydrolytic water molecule or the side chain of Gln-204. The binding site for RGS4 on G(i alpha1) is also consistent with the activity of RGS proteins as antagonists of G(alpha) effectors.

Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP.

We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides. BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM.

Structure of the first C2 domain of synaptotagmin I: A novel Ca2+/phospholipid-binding fold

C2 domains are regulatory sequence motifs that occur widely in nature. Synaptotagmin I, a synaptic vesicle protein involved in the Ca2+ regulation of exocytosis, contains two C2 domains, the first of which acts as a Ca2+ sensor. We now describe the three-dimensional structure of this C2 domain at 1.9 A resolution in both the Ca(2+)-bound and Ca(2+)-free forms. The C2 polypeptide forms an eight-stranded beta sandwich constructed around a conserved four-stranded motif designated as a C2 key. Ca2+ binds in a cup-shaped depression between two polypeptide loops located at the N- and C-termini of the C2-key motif.

Tertiary and quaternary structural changes in Giα1 induced by GTP hydrolysis

Crystallographic analysis of 2.2 angstrom resolution shows that guanosine triphosphate (GTP) hydrolysis triggers conformational changes in the heterotrimeric G-protein α subunit, Giα1. The switch II and switch III segments become disordered, and linker II connecting the Ras and α helical domains moves, thus altering the structures of potential effector and βγ binding regions. Contacts between the α-helical and Ras domains are weakened, possibly facilitating the release of guanosine diphosphate (GDP). The amino and carboxyl termini, which contain receptor and βγ binding determinants, are disordered in the complex with GTP, but are organized into a compact microdomain on GDP hydrolysis. The amino terminus also forms extensive quaternary contacts with neighboring α subunits in the lattice, suggesting that multimers of α subunits or heterotrimers may play a role in signal transduction.

Modularity in the TNF.receptor family

Tumour necrosis factor (TNF) receptor family members regulate processes that range from cell proliferation to programmed cell death. The extracellular, ligand-binding domains of these proteins consist of small, cysteine-rich subdomains, first observed in the three-dimensional structures of the type I TNF receptor. A structure-based alignment of TNFR family members indicates that the extracellular domains are constructed primarily of two small polypeptide modules. These modules play distinctive structural roles in the architecture of the domains. Analogues of at least one of these modules can be found in the domains of other receptors and extracellular proteins. Variations in their sequence and order of assembly are expected to account for differences in shape, flexibility and ligand specificity.

Structural changes in glycogen phosphorylase induced by phosphorylation

A comparison of the refined crystal structures of dimeric glycogen phosphorylase b and a reveals structural changes that represent the first step in the activation of the enzyme. On phosphorylation of serine-14, the N-terminus of each subunit assumes an ordered helical conformation and binds to the surface of the dimer. The consequent structural changes at the N- and C-terminal regions lead to strengthened interactions between subunits and alter the binding sites for allosteric effectors and substrates.

Exchange of Substrate and Inhibitor Specificities between Adenylyl and Guanylyl Cyclases

The active sites of guanylyl and adenylyl cyclases are closely related. The crystal structure of adenylyl cyclase and modeling studies suggest that specificity for ATP or GTP is dictated in part by a few amino acid residues, invariant in each family, that interact with the purine ring of the substrate. By exchanging these residues between guanylyl cyclase and adenylyl cyclase, we can completely change the nucleotide specificity of guanylyl cyclase and convert adenylyl cyclase into a nonselective purine nucleotide cyclase. The activities of these mutant enzymes remain fully responsive to their respective stimulators, sodium nitroprusside and Gsα. The specificity of nucleotide inhibitors of guanylyl and adenylyl cyclases that do not act competitively with respect to substrate are similarly altered, indicative of their action at the active sites of these enzymes.

Cytokine structural taxonomy and mechanisms of receptor engagement: Current opinion in structural biology 1993, 3:815–827

Abstract Seven discrete families of cytokines have been identified by sequence and structural analysis. Within this diverse set of protein folds, the hematopoietic growth factors and tumor necrosis factor-like cytokines display a remarkable degree of variation upon their respective topological frameworks. In addition, prototype X-ray structures of ligand-receptor complexes reveal two different strategies of receptor engagement.

Three-Dimensional Structure of a Complex between the Death Domains of Pelle and Tube

The interaction of the serine/threonine kinase Pelle and adaptor protein Tube through their N-terminal death domains leads to the nuclear translocation of the transcription factor Dorsal and activation of zygotic patterning genes during Drosophila embryogenesis. Crystal structure of the Pelle and Tube death domain heterodimer reveals that the two death domains adopt a six-helix bundle fold and are arranged in an open-ended linear array with plastic interfaces mediating their interactions. The Tube death domain has an insertion between helices 2 and 3, and a C-terminal tail making significant and indispensable contacts in the heterodimer. In vivo assays of Pelle and Tube mutants confirmed that the integrity of the major heterodimer interface is critical to the activity of these molecules.