Structure and function in rhodopsin: Topology of the C-terminal polypeptide chain in relation to the cytoplasmic loops*

Cysteine mutagenesis and site-directed spin labeling in the C-terminal region of rhodopsin have been used to probe the local structure and proximity of that region to the cytoplasmic loops. Each of the native amino acids in the sequence T335–T340 was replaced with Cys, one at a time. The sulfhydryl groups of all mutants reacted rapidly with the sulfhydryl reagent 4,4′-dithiodipyridine, which indicated a high degree of solvent accessibility. Furthermore, to probe the proximity relationships, a series of double Cys mutants was constructed. One Cys in all sets was at position 338 and the other was at a position in the sequence S240–V250 in the EF interhelical loop, at position 65 in the AB interhelical loop, or at position 140 in the CD interhelical loop. In the dark state, no significant disulfide formation was observed between C338 and C65 or C140 under the conditions used, whereas a relatively rapid disulfide formation was observed between C338 and C242 or C245. Spin labels in the double Cys mutants showed the strongest magnetic interactions between the nitroxides attached to C338 and C245 or C246. Light activation of the double mutant T242C/S338C resulted in slower disulfide formation, whereas interactions between nitroxides at C338 and C245 or C246 decreased. These results suggest the proximity of the C-terminal residue C338 to residues located on the outer face of a cytoplasmic helical extension of the F helix with an apparent increase of distance upon photoactivation.

Transition-state structure as a unifying basis in protein-folding mechanisms: Contact order, chain topology, stability, and the extended nucleus mechanism

I attempt to reconcile apparently conflicting factors and mechanisms that have been proposed to determine the rate constant for two-state folding of small proteins, on the basis of general features of the structures of transition states. Φ-Value analysis implies a transition state for folding that resembles an expanded and distorted native structure, which is built around an extended nucleus. The nucleus is composed predominantly of elements of partly or well-formed native secondary structure that are stabilized by local and long-range tertiary interactions. These long-range interactions give rise to connecting loops, frequently containing the native loops that are poorly structured. I derive an equation that relates differences in the contact order of a protein to changes in the length of linking loops, which, in turn, is directly related to the unfavorable free energy of the loops in the transition state. Kinetic data on loop extension mutants of CI2 and α-spectrin SH3 domain fit the equation qualitatively. The rate of folding depends primarily on the interactions that directly stabilize the nucleus, especially those in native-like secondary structure and those resulting from the entropy loss from the connecting loops, which vary with contact order. This partitioning of energy accounts for the success of some algorithms that predict folding rates, because they use these principles either explicitly or implicitly. The extended nucleus model thus unifies the observations of rate depending on both stability and topology.

Crystal structure of the HLA-Cw3 allotype-specific killer cell inhibitory receptor KIR2DL2

Killer cell inhibitory receptors (KIR) protect class I HLAs expressing target cells from natural killer (NK) cell-mediated lysis. To understand the molecular basis of this receptor-ligand recognition, we have crystallized the extracellular ligand-binding domains of KIR2DL2, a member of the Ig superfamily receptors that recognize HLA-Cw1, 3, 7, and 8 allotypes. The structure was determined in two different crystal forms, an orthorhombic P212121 and a trigonal P3221 space group, to resolutions of 3.0 and 2.9 Å, respectively. The overall fold of this structure, like KIR2DL1, exhibits K-type Ig topology with cis-proline residues in both domains that define β-strand switching, which sets KIR apart from the C2-type hematopoietic growth hormone receptor fold. The hinge angle of KIR2DL2 is approximately 80°, 14° larger than that observed in KIR2DL1 despite the existence of conserved hydrophobic residues near the hinge region. There is also a 5° difference in the observed hinge angles in two crystal forms of 2DL2, suggesting that the interdomain hinge angle is not fixed. The putative ligand-binding site is formed by residues from several variable loops with charge distribution apparently complementary to that of HLA-C. The packing of the receptors in the orthorhombic crystal form offers an intriguing model for receptor aggregation on the cell surface.

Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1α, a potent ligand for the HIV-1 “fusin” coreceptor

Stromal cell-derived factor-1α (SDF-1α ) is a member of the chemokine superfamily and functions as a growth factor and chemoattractant through activation of CXCR4/LESTR/Fusin, a G protein-coupled receptor. This receptor also functions as a coreceptor for T-tropic syncytium-inducing strains of HIV-1. SDF-1α antagonizes infectivity of these strains by competing with gp120 for binding to the receptor. The crystal structure of a variant SDF-1α ([N33A]SDF-1α ) prepared by total chemical synthesis has been refined to 2.2-Å resolution. Although SDF-1α adopts a typical chemokine β-β-β-α topology, the packing of the α-helix against the β-sheet is strikingly different. Comparison of SDF-1α with other chemokine structures confirms the hypothesis that SDF-1α may be either an ancestral protein from which all other chemokines evolved or the chemokine that is the least divergent from a primordial chemokine. The structure of SDF-1α reveals a positively charged surface ideal for binding to the negatively charged extracellular loops of the CXCR4 HIV-1 coreceptor. This ionic complementarity is likely to promote the interaction of the mobile N-terminal segment of SDF-1α with interhelical sites of the receptor, resulting in a biological response.

Structure of a soluble secreted chemokine inhibitor vCCI (p35) from cowpox virus

Most poxviruses, including variola, the causative agent of smallpox, express a secreted protein of 35 kDa, vCCI, which binds CC-chemokines with high affinity. This viral protein competes with the host cellular CC-chemokine receptors (CCRs), reducing inflammation and interfering with the host immune response. Such proteins or derivatives may have therapeutic uses as anti-inflammatory agents. We have determined the crystal structure to 1.85-Å resolution of vCCI from cowpox virus, the prototype of this poxvirus virulence factor. The molecule is a β-sandwich of topology not previously described. A patch of conserved residues on the exposed face of a β-sheet that is strongly negatively charged might have a role in binding of CC-chemokines, which are positively charged.

Structure of the thrombin complex with triabin, a lipocalin-like exosite-binding inhibitor derived from a triatomine bug

Triabin, a 142-residue protein from the saliva of the blood-sucking triatomine bug Triatoma pallidipennis, is a potent and selective thrombin inhibitor. Its stoichiometric complex with bovine α-thrombin was crystallized, and its crystal structure was solved by Patterson search methods and refined at 2.6-Å resolution to an R value of 0.184. The analysis revealed that triabin is a compact one-domain molecule essentially consisting of an eight-stranded β-barrel. The eight strands A to H are arranged in the order A-C-B-D-E-F-G-H, with the first four strands exhibiting a hitherto unobserved up-up-down-down topology. Except for the B-C inversion, the triabin fold exhibits the regular up-and-down topology of lipocalins. In contrast to the typical ligand-binding lipocalins, however, the triabin barrel encloses a hydrophobic core intersected by a unique salt-bridge cluster. Triabin interacts with thrombin exclusively via its fibrinogen-recognition exosite. Surprisingly, most of the interface interactions are hydrophobic. A prominent exception represents thrombin’s Arg-77A side chain, which extends into a hydrophobic triabin pocket forming partially buried salt bridges with Glu-128 and Asp-135 of the inhibitor. The fully accessible active site of thrombin in this complex is in agreement with its retained hydrolytic activity toward small chromogenic substrates. Impairment of thrombin’s fibrinogen converting activity or of its thrombomodulin-mediated protein C activation capacity upon triabin binding is explained by usage of overlapping interaction sites of fibrinogen, thrombomodulin, and triabin on thrombin. These data demonstrate that triabin inhibits thrombin via a novel and unique mechanism that might be of interest in the context of potential therapeutic applications.

The solution structure of the N-terminal domain of α2-macroglobulin receptor-associated protein

The three-dimensional structure of the N-terminal domain (residues 18–112) of α2-macroglobulin receptor-associated protein (RAP) has been determined by NMR spectroscopy. The structure consists of three helices composed of residues 23–34, 39–65, and 73–88. The three helices are arranged in an up-down-up antiparallel topology. The C-terminal 20 residues were shown not to be in a well defined conformation. A structural model for the binding of RAP to the family of low-density lipoprotein receptors is proposed. It defines a role in binding for both the unordered C terminus and the structural scaffold of the core structure. Pathogenic epitopes for the rat disease Heymann nephritis, an experimental model of human membranous glomerulonephritis, have been identified in RAP and in the large endocytic receptor gp330/megalin. Here we provide the three-dimensional structure of the pathogenic epitope in RAP. The amino acid residues known to form the epitope are in a helix–loop–helix conformation, and from the structure it is possible to rationalize the published results obtained from studies of fragments of the N-terminal domain.

Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase

The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding. Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 Å resolution; it has a parallel α-β topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related helicases. Using data to 2.8 Å resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a “dumbbell” structure consisting of two compact domains connected by an extended linker. By using the structures of other helicases as a template, compact structures can be modeled for eIF4A that suggest (i) helicase motif IV binds RNA; (ii) Arg-298, which is conserved in the DEA(D/H)-box RNA helicase family but is absent from many other helicases, also binds RNA; and (iii) motifs V and VI “link” the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D/H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding.

Crystal structure of a DEAD box protein from the hyperthermophile Methanococcus jannaschii

We have determined the structure of a DEAD box putative RNA helicase from the hyperthermophile Methanococcus jannaschii. Like other helicases, the protein contains two α/β domains, each with a recA-like topology. Unlike other helicases, the protein exists as a dimer in the crystal. Through an interaction that resembles the dimer interface of insulin, the amino-terminal domain's 7-strand β-sheet is extended to 14 strands across the two molecules. Motifs conserved in the DEAD box family cluster in the cleft between domains, and many of their functions can be deduced by mutational data and by comparison with other helicase structures. Several lines of evidence suggest that motif III Ser-Ala-Thr may be involved in binding RNA.

Solution structure of the antiapoptotic protein bcl-2

The structures of two isoforms of Bcl-2 that differ by two amino acids have been determined by NMR spectroscopy. Because wild-type Bcl-2 behaved poorly in solution, the structures were determined by using Bcl-2/Bcl-xL chimeras in which part of the putative unstructured loop of Bcl-2 was replaced with a shortened loop from Bcl-xL. These chimeric proteins have a low pI compared with the wild-type protein and are soluble. The structures of the two Bcl-2 isoforms consist of 6 α-helices with a hydrophobic groove on the surface similar to that observed for the homologous protein, Bcl-xL. Comparison of the Bcl-2 structures to that of Bcl-xL shows that although the overall fold is the same, there are differences in the structural topology and electrostatic potential of the binding groove. Although the structures of the two isoforms of Bcl-2 are virtually identical, differences were observed in the ability of the proteins to bind to a 25-residue peptide from the proapoptotic Bad protein and a 16-residue peptide from the proapoptotic Bak protein. These results suggest that there are subtle differences in the hydrophobic binding groove in Bcl-2 that may translate into differences in antiapoptotic activity for the two isoforms.

Crystal structure of the ectodomain of Methuselah, a Drosophila G protein-coupled receptor associated with extended lifespan

The Drosophila mutant methuselah (mth) was identified from a screen for single gene mutations that extended average lifespan. Mth mutants have a 35% increase in average lifespan and increased resistance to several forms of stress, including heat, starvation, and oxidative damage. The protein affected by this mutation is related to G protein-coupled receptors of the secretin receptor family. Mth, like secretin receptor family members, has a large N-terminal ectodomain, which may constitute the ligand binding site. Here we report the 2.3-Å resolution crystal structure of the Mth extracellular region, revealing a folding topology in which three primarily β-structure-containing domains meet to form a shallow interdomain groove containing a solvent-exposed tryptophan that may represent a ligand binding site. The Mth structure is analyzed in relation to predicted Mth homologs and potential ligand binding features.

Crystal structure of phage P22 tailspike protein complexed with Salmonella sp. O-antigen receptors.

The O-antigenic repeating units of lipopolysaccharides from Salmonella serogroups A, B, and D1 serve as receptors for the phage P22 tailspike protein, which also has receptor destroying endoglycosidase (endorhamnosidase) activity, integrating the functions of both hemagglutinin and neuraminidase in influenza virus. Crystal structures of the tailspike protein in complex with oligosaccharides, comprising two O-antigenic repeating units from Salmonella typhimurium, Salmonella enteritidis, and Salmonella typhi 253Ty were determined at 1.8 A resolution. The active-site topology with Asp-392, Asp-395, and Glu-359 as catalytic residues was identified. Kinetics of binding and cleavage suggest a role of the receptor destroying endorhamnosidase activity primarily for detachment of newly assembled phages.

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

The crystal structure of the immunity protein of colicin E7 suggests a possible colicin-interacting surface.

The immunity protein of colicin E7 (ImmE7) can bind specifically to the DNase-type colicin E7 and inhibit its bactericidal activity. Here we report the 1.8-angstrom crystal structure of the ImmE7 protein. This is the first x-ray structure determined in the superfamily of colicin immunity proteins. The ImmE7 protein consists of four antiparallel alpha-helices, folded in a topology similar to the architecture of a four-helix bundle structure. A region rich in acidic residues is identified. This negatively charged area has the greatest variability within the family of DNase-type immunity proteins; thus, it seems likely that this area is involved in specific binding to colicin. Based on structural, genetic, and kinetic data, we suggest that all the DNase-type immunity proteins, as well as colicins, share a "homologous-structural framework" and that specific interaction between a colicin and its cognate immunity protein relies upon how well these two proteins' charged residues match on the interaction surface, thus leading to specific immunity of the colicin.

The crystal structure of human glycosylation-inhibiting factor is a trimeric barrel with three 6-stranded beta-sheets.

Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis. The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method. The structure was refined to an R factor of 0.168 at 1.9 angstrom resolution. The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded beta-sheets on the inside and six alpha-helices on the outside. There is a 5-angstrom-diameter "hole" through the middle of the barrel. The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor. Each subunit has a new class of alpha + beta sandwich structure consisting of two beta-alpha-beta motifs. These beta-alpha-beta motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different.