Structural mimicry of a native protein by a minimized binding domain

The affinity between molecules depends both on the nature and presentation of the contacts. Here, we observe coupling of functional and structural elements when a protein binding domain is evolved to a smaller functional mimic. Previously, a 38-residue form of the 59-residue B-domain of protein A, termed Z38, was selected by phage display. Z38 contains 13 mutations and binds IgG only 10-fold weaker than the native B-domain. We present the solution structure of Z38 and show that it adopts a tertiary structure remarkably similar to that observed for the first two helices of B-domain in the B-domain/Fc complex [Deisenhofer, J. (1981) Biochemistry 20, 2361–2370], although it is significantly less stable. Based on this structure, we have improved on Z38 by designing a 34-residue disulfide-bonded variant (Z34C) that has dramatically enhanced stability and binds IgG with 9-fold higher affinity. The improved stability of Z34C led to NMR spectra with much greater chemical shift dispersion, resulting in a more precisely determined structure. Z34C, like Z38, has a structure virtually identical to the equivalent region from native protein A domains. The well-defined hydrophobic core of Z34C reveals key structural features that have evolved in this small, functional domain. Thus, the stabilized two-helix peptide, about half the size and having one-third of the remaining residues altered, accurately mimics both the structure and function of the native domain.

Structure of the imprinted mouse Snrpn gene and establishment of its parental-specific methylation pattern

The mouse Snrpn gene encodes the Smn protein, which is involved in RNA splicing. The gene maps to a region in the central part of chromosome 7 that is syntenic to the Prader–Willi/Angelman syndromes (PWS-AS) region on human chromosome 15q11-q13. The mouse gene, like its human counterpart, is imprinted and paternally expressed, primarily in brain and heart. We provide here a detailed description of the structural features and differential methylation pattern of the gene. We have identified a maternally methylated region at the 5′ end (DMR1), which correlates inversely with the Snrpn paternal expression. We also describe a region at the 3′ end of the gene (DMR2) that is preferentially methylated on the paternal allele. Analysis of Snrpn mRNA levels in a methylase-deficient mouse embryo revealed that maternal methylation of DMR1 may play a role in silencing the maternal allele. Yet both regions, DMR1 and DMR2, inherit the parental-specific methylation profile from the gametes. This methylation pattern is erased in 12.5-days postcoitum (dpc) primordial germ cells and reestablished during gametogenesis. DMR1 is remethylated during oogenesis, whereas DMR2 is remethylated during spermatogenesis. Once established, these methylation patterns are transmitted to the embryo and maintained, protected from methylation changes during embryogenesis and cell differentiation. Transfections of DMR1 and DMR2 into embryonic stem cells and injection into pronuclei of fertilized eggs reveal that embryonic cells lack the capacity to establish anew the differential methylation pattern of Snrpn. That all PWS patients lack DMR1, together with the overall high resemblance of the mouse gene to the human SNRPN, offers an excellent experimental tool to study the regional control of this imprinted chromosomal domain.

Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody FV fragment

The aa3 type cytochrome c oxidase consisting of the core subunits I and II only was isolated from the soil bacterium Paracoccus denitrificans and crystallized as complex with a monoclonal antibody Fv fragment. Crystals could be grown in the presence of a number of different nonionic detergents. However, only undecyl-β-d-maltoside and cyclohexyl-hexyl-β-d-maltoside yielded well-ordered crystals suitable for high resolution x-ray crystallographic studies. The crystals belong to space group P212121 and diffract x-rays to at least 2.5 Å (1 Å = 0.1 nm) resolution using synchrotron radiation. The structure was determined to a resolution of 2.7 Å using molecular replacement and refined to a crystallographic R-factor of 20.5% (Rfree = 25.9%). The refined model includes subunits I and II and the 2 chains of the Fv fragment, 2 heme A molecules, 3 copper atoms, and 1 Mg/Mn atom, a new metal (Ca) binding site, 52 tentatively identified water molecules, and 9 detergent molecules. Only four of the water molecules are located in the cytoplasmic half of cytochrome c oxidase. Most of them are near the interface of subunits I and II. Several waters form a hydrogen-bonded cluster, including the heme propionates and the Mg/Mn binding site. The Fv fragment binds to the periplasmic polar domain of subunit II and is critically involved in the formation of the crystal lattice. The crystallization procedure is well reproducible and will allow for the analysis of the structures of mechanistically interesting mutant cytochrome c oxidases.

The cysteine-rich frizzled domain of Frzb-1 is required and sufficient for modulation of Wnt signaling

Convincing evidence has accumulated to identify the Frizzled proteins as receptors for the Wnt growth factors. In parallel, a number of secreted frizzled-like proteins with a conserved N-terminal frizzled motif have been identified. One of these proteins, Frzb-1, binds Wnt-1 and Xwnt-8 proteins and antagonizes Xwnt-8 signaling in Xenopus embryos. Here we report that Frzb-1 blocks Wnt-1 induced cytosolic accumulation of β-catenin, a key component of the Wnt signaling pathway, in human embryonic kidney cells. Structure/function analysis reveals that complete removal of the frizzled domain of Frzb-1 abolishes the Wnt-1/Frzb-1 protein interaction and the inhibition of Wnt-1 mediated axis duplication in Xenopus embryos. In contrast, removal of the C-terminal portion of the molecule preserves both Frzb-Wnt binding and functional inhibition of Wnt signaling. Partial deletions of the Frzb-1 cysteine-rich domain maintain Wnt-1 interaction, but functional inhibition is lost. Taken together, these findings support the conclusion that the frizzled domain is necessary and sufficient for both activities. Interestingly, Frzb-1 does not block Wnt-5A signaling in a Xenopus functional assay, even though Wnt-5A coimmunoprecipitates with Frzb-1, suggesting that coimmunoprecipitation does not necessarily imply inhibition of Wnt function.

The signal recognition particle receptor of Escherichia coli (FtsY) has a nucleotide exchange factor built into the GTPase domain

Targeting of many secretory and membrane proteins to the inner membrane in Escherichia coli is achieved by the signal recognition particle (SRP) and its receptor (FtsY). In E. coli SRP consists of only one polypeptide (Ffh), and a 4.5S RNA. Ffh and FtsY each contain a conserved GTPase domain (G domain) with an α-helical domain on its N terminus (N domain). The nucleotide binding kinetics of the NG domain of the SRP receptor FtsY have been investigated, using different fluorescence techniques. Methods to describe the reaction kinetically are presented. The kinetics of interaction of FtsY with guanine nucleotides are quantitatively different from those of other GTPases. The intrinsic guanine nucleotide dissociation rates of FtsY are about 105 times higher than in Ras, but similar to those seen in GTPases in the presence of an exchange factor. Therefore, the data presented here show that the NG domain of FtsY resembles a GTPase–nucleotide exchange factor complex not only in its structure but also kinetically. The I-box, an insertion present in all SRP-type GTPases, is likely to act as an intrinsic exchange factor. From this we conclude that the details of the GTPase cycle of FtsY and presumably other SRP-type GTPases are fundamentally different from those of other GTPases.

CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis

We have identified and characterized CLARP, a caspase-like apoptosis-regulatory protein. Sequence analysis revealed that human CLARP contains two amino-terminal death effector domains fused to a carboxyl-terminal caspase-like domain. The structure and amino acid sequence of CLARP resemble those of caspase-8, caspase-10, and DCP2, a Drosophila melanogaster protein identified in this study. Unlike caspase-8, caspase-10, and DCP2, however, two important residues predicted to be involved in catalysis were lost in the caspase-like domain of CLARP. Analysis with fluorogenic substrates for caspase activity confirmed that CLARP is catalytically inactive. CLARP was found to interact with caspase-8 but not with FADD/MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathway. Expression of CLARP induced apoptosis, which was blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk). Moreover, CLARP augmented the killing ability of caspase-8 and FADD/MORT-1 in mammalian cells. The human clarp gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis.

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 helical domain of a G protein α subunit is a regulator of its effector

The α subunit (Gα) of heterotrimeric G proteins is a major determinant of signaling selectivity. The Gα structure essentially comprises a GTPase “Ras-like” domain (RasD) and a unique α-helical domain (HD). We used the vertebrate phototransduction model to test for potential functions of HD and found that the HD of the retinal transducin Gα (Gαt) and the closely related gustducin (Gαg), but not Gαi1, Gαs, or Gαq synergistically enhance guanosine 5′-γ[-thio]triphosphate bound Gαt (GαtGTPγS) activation of bovine rod cGMP phosphodiesterase (PDE). In addition, both HDt and HDg, but not HDi1, HDs, or HDq attenuate the trypsin-activated PDE. GαtGDP and HDt attenuation of trypsin-activated PDE saturate with similar affinities and to an identical 38% of initial activity. These data suggest that interaction of intact Gαt with the PDE catalytic core may be caused by the HD moiety, and they indicate an independent site(s) for the HD moiety of Gαt within the PDE catalytic core in addition to the sites for the inhibitory Pγ subunits. The HD moiety of GαtGDP is an attenuator of the activated catalytic core, whereas in the presence of activated GαtGTPγS the independently expressed HDt is a potent synergist. Rhodopsin catalysis of Gαt activation enhances the PDE activation produced by subsaturating levels of Gαt, suggesting a HD-moiety synergism from a transient conformation of Gαt. These results establish HD-selective regulations of vertebrate retinal PDE, and they provide evidence demonstrating that the HD is a modulatory domain. We suggest that the HD works in concert with the RasD, enhancing the efficiency of G protein signaling.

Prion protein NMR structure and species barrier for prion diseases

The structural basis of species specificity of transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy or “mad cow disease” and Creutzfeldt–Jakob disease in humans, has been investigated using the refined NMR structure of the C-terminal domain of the mouse prion protein with residues 121–231. A database search for mammalian prion proteins yielded 23 different sequences for the fragment 124–226, which display a high degree of sequence identity and show relevant amino acid substitutions in only 18 of the 103 positions. Except for a unique isolated negative surface charge in the bovine protein, the amino acid differences are clustered in three distinct regions of the three-dimensional structure of the cellular form of the prion protein. Two of these regions represent potential species-dependent surface recognition sites for protein–protein interactions, which have independently been implicated from in vitro and in vivo studies of prion protein transformation. The third region consists of a cluster of interior hydrophobic side chains that may affect prion protein transformation at later stages, after initial conformational changes in the cellular protein.

Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus

We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 2.8-Å resolution. This enzyme is a key target for developing specific antiviral therapy. The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains. The fingers subdomain contains a region, the “fingertips,” that shares the same fold with reverse transcriptases. Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent. In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998) Science 282, 1669–1675]. This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP. It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds.

Neuronal type information encoded in the basic-helix–loop–helix domain of proneural genes

The proneural genes encode basic-helix–loop–helix (bHLH) proteins and promote the formation of distinct types of sensory organs. In Drosophila, two sets of proneural genes, atonal (ato) and members of the achaete–scute complex (ASC), are required for the formation of chordotonal (ch) organs and external sensory (es) organs, respectively. We assayed the production of sensory organs in transgenic flies expressing chimeric genes of ato and scute (sc), a member of ASC, and found that the information that specifies ch organs resides in the bHLH domain of ato; chimeras containing the b domain of ato and the HLH domain of sc also induced ch organ formation, but to a lesser extent than those containing the bHLH domain of ato. The b domains of ato and sc differ in seven residues. Mutations of these seven residues in the b domain of ato suggest that most or perhaps all of these residues are required for induction of ch organs. None of these seven residues is predicted to contact DNA directly by computer simulation using the structure of the myogenic factor MyoD as a model, implying that interaction of ato with other cofactors is likely to be involved in neuronal type specification.

Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation

Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyΔC1225 and MyΔC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-β and ori-γ) being somewhat preferred. We show here that the chromatin in the ori-β and ori-γ regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G1/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.

Crystal structure of glutamate-1-semialdehyde aminomutase: An α2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity

The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC 5.4.3.8), an α2-dimeric enzyme from Synechococcus, has been determined by x-ray crystallography using heavy atom derivative phasing. The structure, refined at 2.4-Å resolution to an R-factor of 18.7% and good stereochemistry, explains many of the enzyme’s unusual specificity and functional properties. The overall fold is that of aspartate aminotransferase and related B6 enzymes, but it also has specific features. The structure of the complex with gabaculine, a substrate analogue, shows unexpectedly that the substrate binding site involves residues from the N-terminal domain of the molecule, notably Arg-32. Glu-406 is suitably positioned to repel α-carboxylic acids, thereby suggesting a basis for the enzyme’s reaction specificity. The subunits show asymmetry in cofactor binding and in the mobilities of the residues 153–181. In the unliganded enzyme, one subunit has the cofactor bound as an aldimine of pyridoxal phosphate with Lys-273 and, in this subunit, residues 153–181 are disordered. In the other subunit in which the cofactor is not covalently bound, residues 153–181 are well defined. Consistent with the crystallographically demonstrated asymmetry, a form of the enzyme in which both subunits have pyridoxal phosphate bound to Lys-273 through a Schiff base showed biphasic reduction by borohydride in solution. Analysis of absorption spectra during reduction provided evidence of communication between the subunits. The crystal structure of the reduced form of the enzyme shows that, despite identical cofactor binding in each monomer, the structural asymmetry at residues 153–181 remains.

The α1 domain of HLA-G1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: Is HLA-G the public ligand for natural killer cell inhibitory receptors?

We have investigated the protective role of the membrane-bound HLA-G1 and HLA-G2 isoforms against natural killer (NK) cell cytotoxicity. For this purpose, HLA-G1 and HLA-G2 cDNAs were transfected into the HLA class I-negative human K562 cell line, a known reference target for NK lysis. The HLA-G1 protein, encoded by a full-length mRNA, presents a structure similar to that of classical HLA class I antigens. The HLA-G2 protein, deduced from an alternatively spliced transcript, consists of the α1 domain linked to the α3 domain. In this study we demonstrate that (i) HLA-G2 is present at the cell surface as a truncated class I molecule associated with β2-microglobulin; (ii) NK cytolysis, observed in peripheral blood mononuclear cells and in polyclonal CD3− CD16+ CD56+ NK cells obtained from 20 donors, is inhibited by both HLA-G1 and HLA-G2; this HLA-G-mediated inhibition is reversed by blocking HLA-G with a specific mAb; this led us to the conjecture that HLA-G is the public ligand for NK inhibitory receptors (NKIR) present in all individuals; (iii) the α1 domain common to HLA-G1 and HLA-G2 could mediate this protection from NK lysis; and (iv) when transfected into the K562 cell line, both HLA-G1 and HLA-G2 abolish lysis by the T cell leukemia NK-like YT2C2 clone due to interaction between the HLA-G isoform on the target cell surface and a membrane receptor on YT2C2. Because NKIR1 and NKIR2, known to interact with HLA-G, were undetectable on YT2C2, we conclude that a yet-unknown specific receptor for HLA-G1 and HLA-G2 is present on these cells.