The crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 reveals novel dimerization motif for Hsp40

The molecular chaperone Hsp40 functions as a dimer. The dimer formation is critical for Hsp40 molecular chaperone activity to facilitate Hsp70 to refold non-native polypeptides. We have determined the crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 that is responsible for Ydj1 dimerization by MAD method. The C-terminal fragment of Ydj1 comprises of the domain III of Ydj1 and the Ydj1 C-terminal dimerization motif. The crystal structure indicates that the dimerization motif of type I Hsp40 Ydj1 differs significantly from that of yeast type II Hsp40. The C terminus of type I Hsp40 Ydj1 from one monomer forms beta-strands with the domain III from the other monomer in the homo-dimer. The L372 from Ydj1 C terminus inserts its side-chain into a hydrophobic pocket on domain III. The modeled full-length Ydj1 dimer structure reveals that a large cleft is formed between the two monomers. The domain IIs of Ydj1 monomers that contain the zinc-finger motifs points directly against each other.     

Cooperative binding of the hnRNP K three KH domains to mRNA targets

The heterogeneous nuclear ribonucleoprotein (hnRNP) K homology (KH) domain is an evolutionarily conserved module that binds short ribonucleotide sequences. KH domains most often are present in multiple copies per protein. In vitro studies of hnRNP K and other KH domain bearing proteins have yielded conflicting results regarding the relative contribution of each KH domain to the binding of target RNAs. To assess this RNA-binding we used full-length hnRNP K, its fragments and the yeast ortholog as baits in the yeast three-hybrid system. The results demonstrate that in this heterologous in vivo system, the three KH domains bind RNA synergistically and that a single KH domain, in comparison, binds RNA weakly.     

The non-canonical hydroxylase structure of YfcM reveals a metal ion-coordination motif required for EF-P hydroxylation

EF-P is a bacterial tRNA-mimic protein, which accelerates the ribosome-catalyzed polymerization of poly-prolines. In Escherichia coli, EF-P is post-translationally modified on a conserved lysine residue. The post-translational modification is performed in a two-step reaction involving the addition of a β-lysine moiety and the subsequent hydroxylation, catalyzed by PoxA and YfcM, respectively. The β-lysine moiety was previously shown to enhance the rate of poly-proline synthesis, but the role of the hydroxylation is poorly understood. We solved the crystal structure of YfcM and performed functional analyses to determine the hydroxylation mechanism. In addition, YfcM appears to be structurally distinct from any other hydroxylase structures reported so far. The structure of YfcM is similar to that of the ribonuclease YbeY, even though they do not share sequence homology. Furthermore, YfcM has a metal ion-coordinating motif, similar to YbeY. The metal ion-coordinating motif of YfcM resembles a 2-His-1-carboxylate motif, which coordinates an Fe(II) ion and forms the catalytic site of non-heme iron enzymes. Our findings showed that the metal ion-coordinating motif of YfcM plays an essential role in the hydroxylation of the β-lysylated lysine residue of EF-P. Taken together, our results suggested the potential catalytic mechanism of hydroxylation by YfcM.     

The structures of exocyst subunit Exo70p and the Exo84p C-terminal domains reveal a common motif

The exocyst is a large complex that is required for tethering vesicles at the final stages of the exocytic pathway in all eukaryotes. Here we present the structures of the Exo70p subunit of this complex and of the C-terminal domains of Exo84p, at 2.0-A and 2.85-A resolution, respectively. Exo70p forms a 160-A-long rod with a novel fold composed of contiguous alpha-helical bundles. The Exo84p C terminus also forms a long rod (80 A), which unexpectedly has the same fold as the Exo70p N terminus. Our structural results and our experimental observations concerning the interaction between Exo70p and other exocyst subunits or Rho3p GTPase are consistent with an architecture wherein exocyst subunits are composed of mostly helical modules strung together into long rods.     

NMR structure of the heme chaperone CcmE reveals a novel functional motif

The concept of metal chaperones involves transient binding of metallic cofactors by specific proteins for delivery to enzymes in which they function. Metal chaperones thus provide a protective, as well as a transport, function. We report the first structure of a heme chaperone, CcmE, which comprises these two functions. We propose that the covalent attachment of heme to an exposed histidine occurs after heme binding at the surface of a rigid molecule with a flexible C-terminal domain. CcmE belongs to a family of proteins with a specific fold, which all share a function in delivery of specific molecular cargo.     

The crystal structure of the superoxide dismutase from Helicobacter pylori reveals a structured C-terminal extension

Superoxide dismutases (SODs) are key enzymes for fighting oxidative stress. Helicobacter pylori produces a single SOD (HpSOD) which contains iron. The structure of this antioxidant protein has been determined at 2.4 A resolution. It is a dimer of two identical subunits with one iron ion per monomer. The protein shares 53% sequence identity with the corresponding enzyme from Escherichia coli. The model is compared with those of other dimeric Fe-containing SODs. HpSOD shows significant differences in relation to other SODs, the most important being an extended C-terminal tail. This structure provides a model for closely related sequences from species such as Campylobacter, where no structures are currently known. The structure of extended carboxyl termini is discussed in light of putative functions it may serve.     

Crystal structure of yeast allantoicase reveals a repeated jelly roll motif

Allantoicase (EC 3.5.3.4) catalyzes the conversion of allantoate into ureidoglycolate and urea, one of the final steps in the degradation of purines to urea. The mechanism of most enzymes involved in this pathway, which has been known for a long time, is unknown. In this paper we describe the three-dimensional crystal structure of the yeast allantoicase determined at a resolution of 2.6 A by single anomalous diffraction. This constitutes the first structure for an enzyme of this pathway. The structure reveals a repeated jelly roll beta-sheet motif, also present in proteins of unrelated biochemical function. Allantoicase has a hexameric arrangement in the crystal (dimer of trimers). Analysis of the protein sequence against the structural data reveals the presence of two totally conserved surface patches, one on each jelly roll motif. The hexameric packing concentrates these patches into conserved pockets that probably constitute the active site.     

Crystal structure of the dimeric C-terminal domain of TonB reveals a novel fold

The TonB-dependent complex of Gram-negative bacteria couples the inner membrane proton motive force to the active transport of iron.siderophore and vitamin B(12) across the outer membrane. The structural basis of that process has not been described so far in full detail. The crystal structure of the C-terminal domain of TonB from Escherichia coli has now been solved by multiwavelength anomalous diffraction and refined at 1.55-A resolution, providing the first evidence that this region of TonB (residues 164-239) dimerizes. Moreover, the structure shows a novel architecture that has no structural homologs among any known proteins. The dimer of the C-terminal domain of TonB is cylinder-shaped with a length of 65 A and a diameter of 25 A. Each monomer contains three beta strands and a single alpha helix. The two monomers are intertwined with each other, and all six beta-strands of the dimer make a large antiparallel beta-sheet. We propose a plausible model of binding of TonB to FhuA and FepA, two TonB-dependent outer-membrane receptors.     

The eIF1A solution structure reveals a large RNA-binding surface important for scanning function

The translation initiation factor eIF1A is necessary for directing the 43S preinitiation complex from the 5' end of the mRNA to the initiation codon in a process termed scanning. We have determined the solution structure of human eIF1A, which reveals an oligonucleotide-binding (OB) fold and an additional domain. NMR titration experiments showed that eIF1A binds single-stranded RNA oligonucleotides in a site-specific, but non-sequence-specific manner, hinting at an mRNA interaction rather than specific rRNA or tRNA binding. The RNA binding surface extends over a large area covering the canonical OB fold binding site as well as a groove leading to the second domain. Site-directed mutations at multiple positions along the RNA-binding surface were defective in the ability to properly assemble preinitiation complexes at the AUG codon in vitro.     

The crystal structure of the C-terminal domain of Vps28 reveals a conserved surface required for Vps20 recruitment

The endosomal sorting complex I required for transport (ESCRT-I) is composed of the three subunits Vps23/Tsg101, Vps28 and Vps37. ESCRT-I is recruited to cellular membranes during multivesicular endosome biogenesis and by enveloped viruses such as HIV-1 to mediate budding from the cell. Here, we describe the crystal structure of a conserved C-terminal domain from Sacharomyces cerevisiae Vps28 (Vps28-CTD) at 3.05 A resolution which folds independently into a four-helical bundle structure. Co-expression experiments of Vps28-CTD, Vps23 and Vps37 suggest that Vps28-CTD does not directly participate in ESCRT-I assembly and may thus act as an adaptor module for downstream interaction partners. We show through mutagenesis studies that Vps28-CTD employs its strictly conserved surface in the interaction with the ESCRT-III factor Vps20. Furthermore, we present evidence that Vps28-CTD is sufficient to rescue an equine infectious anaemia virus (EIAV) Gag late domain deletion. Vps28-CTD mutations abolishing Vps20 interaction in vitro also prevent the rescue of the EIAV Gag late domain mutant consistent with a potential direct Vps28-ESCRT-III Vps20 recruitment. Therefore, the physiological relevant EIAV Gag-Alix interaction can be functionally replaced by a Gag-Vps28-CTD fusion. Because both Alix and Vps28-CTD can directly recruit ESCRT-III proteins, ESCRT-III assembly coupled to Vps4 action may therefore constitute the minimal budding machinery for EIAV release.     

The solution structure of a tetraheme cytochrome from Shewanella frigidimarina reveals a novel family structural motif

The bacteria belonging to the genus Shewanella are facultative anaerobes that utilize a variety of terminal electron acceptors which includes soluble and insoluble metal oxides. The tetraheme c-type cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 ( Sfc) contains 86 residues and is involved in the Fe(III) reduction pathways. Although the functional properties of Sfc redox centers are quite well described, no structures are available for this protein. In this work, we report the solution structure of the reduced form of Sfc. The overall fold is completely different from those of the tetraheme cytochromes c 3 and instead has similarities with the tetraheme cytochrome recently isolated from Shewanella oneidensis ( Soc). Comparison of the tetraheme cytochromes from Shewanella shows a considerable diversity in their primary structure and heme reduction potentials, yet they have highly conserved heme geometry, as is the case for the family of tetraheme cytochromes isolated from Desulfovibrio spp.     

The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.

Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3' untranslated region (UTR). These AU-rich sequences appear to be key determinants of the short half-lives of these mRNAs, although the sequence features of these elements and the mechanism by which they target mRNAs for rapid decay have not been fully defined. We have examined the features of AU-rich elements (AREs) that are crucial for their function as determinants of mRNA instability in mammalian cells by testing the ability of various mutant c-fos AREs and synthetic AREs to direct rapid mRNA deadenylation and decay when inserted within the 3' UTR of the normally stable beta-globin mRNA. Evidence is presented that the pentamer AUUUA, which previously was suggested to be the minimal determinant of instability present in mammalian AREs, cannot direct rapid mRNA deadenylation and decay. Instead, the nonomer UUAUUUAUU is the elemental AU-rich sequence motif that destabilizes mRNA. Removal of one uridine residue from either end of the nonamer (UUAUUUAU or UAUUUAUU) results in a decrease of potency of the element, while removal of a uridine residue from both ends of the nonamer (UAUUUAU) eliminates detectable destabilizing activity. The inclusion of an additional uridine residue at both ends of the nonamer (UUUAUUUAUUU) does not further increase the efficacy of the element. Taken together, these findings suggest that the nonamer UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA. Additional ARE potency is achieved by combining multiple copies of this nonamer in a single mRNA 3' UTR. Furthermore, analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the UUAUUUAUU motif targets mRNA for rapid deadenylation as an early step in the mRNA decay process.     

NMR structure of the hRap1 Myb motif reveals a canonical three-helix bundle lacking the positive surface charge typical of Myb DNA-binding domains.

Mammalian telomeres are composed of long tandem arrays of double-stranded telomeric TTAGGG repeats associated with the telomeric DNA-binding proteins, TRF1 and TRF2. TRF1 and TRF2 contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In the budding yeast, telomeric DNA is associated with scRap1p, which has a central DNA-binding domain that contains two structurally related Myb domains connected by a long linker, an N-terminal BRCT domain, and a C-terminal RCT domain. Recently, the human ortholog of scRap1p (hRap1) was identified and shown to contain a BRCT domain and an RCT domain similar to scRap1p. However, hRap1 contained only one recognizable Myb motif in the center of the protein. Furthermore, while scRap1p binds telomeric DNA directly, hRap1 has no DNA-binding ability. Instead, hRap1 is tethered to telomeres by TRF2. Here, we have determined the solution structure of the Myb domain of hRap1 by NMR. It contains three helices maintained by a hydrophobic core. The architecture of the hRap1 Myb domain is very close to that of each of the Myb domains from TRF1, scRap1p and c-Myb. However, the electrostatic potential surface of the hRap1 Myb domain is distinguished from that of the other Myb domains. Each of the minimal DNA-binding domains, containing one Myb domain in TRF1 and two Myb domains in scRap1p and c-Myb, exhibits a positively charged broad surface that contacts closely the negatively charged backbone of DNA. By contrast, the hRap1 Myb domain shows no distinct positive surface, explaining its lack of DNA-binding activity. The hRap1 Myb domain may be a member of a second class of Myb motifs that lacks DNA-binding activity but may interact instead with other proteins. Other possible members of this class are the c-Myb R1 Myb domain and the Myb domains of ADA2 and Adf1. Thus, while the folds of all Myb domains resemble each other closely, the function of each Myb domain depends on the amino acid residues that are located on the surface of each protein.     

The structure of the carboxyltransferase component of acetyl-coA carboxylase reveals a zinc-binding motif unique to the bacterial enzyme

Acetyl-coA carboxylase (ACC) is a central metabolic enzyme that catalyzes the committed step in fatty acid biosynthesis: biotin-dependent conversion of acetyl-coA to malonyl-coA. The bacterial carboxyltransferase (CT) subunit of ACC is a target for the design of novel therapeutics that combat severe, hospital-acquired infections resistant to the established classes of frontline antimicrobials. Here, we present the structures of the bacterial CT subunits from two prevalent nosocomial pathogens, Staphylococcus aureus and Escherichia coli, at a resolution of 2.0 and 3.0 A, respectively. Both structures reveal a small, independent zinc-binding domain that lacks a complement in the primary sequence or structure of the eukaryotic homologue.     

The C-terminal domains of TARPs: Unexpectedly versatile domains

AMPA receptors mediate the majority of fast synaptic transmission in the central nervous system and are therefore among the most intensively studied ligand-gated ion channels over the last decades. However, the recent discovery that native AMPA receptor complexes contain auxiliary subunits classified as transmembrane AMPA receptor regulatory proteins (TARPs) was quite a surprise and dramatically changed the field of AMPA receptor research. TARPs regulate trafficking as well as synaptic localization of AMPA receptors, and alter their pharmacological and biophysical properties, generally resulting in strongly elevated receptor-mediated currents. Thus, the association of AMPA receptors with TARPs increases receptor heterogeneity and diversity of postsynaptic currents. In this regard, unravelling the mechanisms by which TARPs modulate AMPA receptor function is an intriguing challenge. Studying the functional importance of the carboxy-terminal domain (CTD) of TARPs for receptor modulation, we found that the increased trafficking mediated by the two TARPs γ2 and γ3 is attributable to their CTDs. Furthermore, we demonstrated that the CTD additionally determines the differences between TARPs regarding their modulation of AMPA receptor function. As a case in point, we showed a unique role of the CTD of γ4, suggesting that TARPs modulate AMPA receptor function via individual mechanisms.     

The solution structure of the C-terminal modular pair from Clostridium perfringens mu-toxin reveals a noncellulosomal dockerin module

The genome of the opportunistic pathogen Clostridium perfringens encodes a large number of secreted glycoside hydrolases. Their predicted activities indicate that they are involved in the breakdown of complex carbohydrates and other glycans found in the mucosal layer of the human gastrointestinal tract, within the extracellular matrix, and on the surface of host cells. One such group of these enzymes is the family 84 glycoside hydrolases, which has predicted hyaluronidase activity and comprises five members [C. perfringens glycoside hydrolase family 84 (CpGH84) A-E]. The first identified member, CpGH84A, corresponds to the μ-toxin whose modular architecture includes an N-terminal catalytic domain, four family 32 carbohydrate-binding modules, three FIVAR modules of unknown function, and a C-terminal putative calcium-binding module. Here, we report the solution NMR structure of the C-terminal modular pair from the μ-toxin. The three-helix bundle FIVAR module displays structural homology to a heparin-binding module within the N-terminal of the a C protein from group B Streptoccocus. The C-terminal module has a typical calcium-binding dockerin fold comprising two anti-parallel helices that form a planar face with EF-hand calcium-binding loops at opposite ends of the module. The size of the helical face of the μ-toxin dockerin module is approximately equal to the planar region recently identified on the surface of a cohesin-like X82 module of CpGH84C. Size-exclusion chromatography and heteronuclear NMR-based chemical shift mapping studies indicate that the helical face of the dockerin module recognizes the CpGH84C X82 module. These studies represent the structural characterization of a noncellulolytic dockerin module and its interaction with a cohesin-like X82 module. Dockerin/X82-mediated enzyme complexes may have important implications in the pathogenic properties of C. perfringens.     

The crystal structure of thymidylate synthase from Pneumocystis carinii reveals a fungal insert important for drug design

Thymidylate synthase from Pneumocystis carinii (PcTS) is an especially important drug target, since P. carinii is a fungus that causes opportunistic pneumonia infections in immune-compromised patients and is among the leading causes of death of AIDS patients. Thymidylate synthase (TS) is the sole enzyme responsible for the de novo production of deoxythymidine monophosphate and hence is crucial for DNA replication in every organism. Inhibitors selective for P. carinii TS over human TS would be greatly beneficial in combating this disease. The crystal structure of TS from P. carinii bound to its substrate, dUMP, and a cofactor mimic, CB3717, was determined to 2.6 A resolution. A comparison with other species of TS shows that the volume of the closed PcTS active-site is 20 % larger than that of five other TS closed active-sites. A two-residue proline insert that is strictly conserved among all fungal species of TS, and a novel C-terminal closing interaction involving a P. carinii-specific tyrosine residue are primarily responsible for this increase in volume. The structure suggests several options for designing an inhibitor specific to PcTS and avoiding interactions with human TS. Taking advantage of the residue substitutions of P. carinii TS over human TS enables the design of a selective inhibitor. Additionally, the larger volume of the active-site of PcTS is an important advantage for designing de novo inhibitors that will exclude the human TS active-site through steric hindrance.     

Structure of the C-terminal RING finger from a RING-IBR-RING/TRIAD motif reveals a novel zinc-binding domain distinct from a RING

The really interesting new gene (RING) family of proteins contains over 400 members with diverse physiological functions. A subset of these domains is found in the context of the RING-IBR-RING/TRIAD motifs which function as E3 ubiquitin ligases. Our sequence analysis of the C-terminal RING (RING2) from this motif show that several metal ligating and hydrophobic residues critical for the formation of a classical RING cross-brace structure are not present. Thus, we determined the structure of the RING2 from the RING-IBR-RING motif of HHARI and showed that RING2 has a completely distinct topology from classical RINGs. Notably, RING2 binds only one zinc atom per monomer rather than two and uses a different hydrophobic network to that of classical RINGs. Additionally, this RING2 topology is novel, bearing slight resemblance to zinc-ribbon motifs around the zinc site and is different from the topologies of the zinc binding sites found in RING and PHDs. We demonstrate that RING2 acts as an E3 ligase in vitro and using mutational analysis deduce the structural features required for this activity. Further, mutations in the RING-IBR-RING of Parkin cause a rare form of Parkinsonism and these studies provide an explanation for those mutations that occur in its RING2. From a comparison of the RING2 structure with those reported for RINGs, we infer sequence determinants that allow discrimination between RING2 and RING domains at the sequence analysis level.