Chaperonin 60: a paradoxical,evolutionarily conserved protein family with multiple moonlighting functions

Chaperonin 60 is the prototypic molecular chaperone, an essential protein in eukaryotes and prokaryotes, whose sequence conservation provides an excellent basis for phylogenetic analysis. Escherichia coli chaperonin 60 (GroEL), the prototype of this family of proteins, has an established oligomeric‐structure‐based folding mechanism and a defined population of folding partners. However, there is a growing number of examples of chaperonin 60 proteins whose crystal structures and oligomeric composition are at variance with GroEL, suggesting that additional complexities in the protein‐folding function of this protein should be expected. In addition, many organisms have multiple chaperonin 60 proteins, some of which have lost their protein‐folding ability. It is emerging that this highly conserved protein has evolved a bewildering variety of additional biological functions – known as moonlighting functions – both within the cell and in the extracellular milieu. Indeed, in some organisms, it is these moonlighting functions that have been left after the loss of the protein‐folding activity. This highlights the major paradox in the biology of chaperonin 60. This article reviews the relationship between the folding and non‐folding (moonlighting) activities of the chaperonin 60 family and discusses current knowledge on their molecular evolution focusing on protein domains involved in the non‐folding chaperonin functions in an attempt to understand the emerging biology of this evolutionarily ancient protein family.     

Solution structure of Vibrio cholerae protein VC0424: a variation of the ferredoxin-like fold

The structure of Vibrio cholerae protein VC0424 was determined by NMR spectroscopy. VC0424 belongs to a conserved family of bacterial proteins of unknown function (COG 3076). The structure has an alpha-beta sandwich architecture consisting of two layers: a four-stranded antiparallel beta-sheet and three side-by-side alpha-helices. The secondary structure elements have the order alphabetaalphabetabetaalphabeta along the sequence. This fold is the same as the ferredoxin-like fold, except with an additional long N-terminal helix, making it a variation on this common motif. A cluster of conserved surface residues on the beta-sheet side of the protein forms a pocket that may be important for the biological function of this conserved family of proteins.     

The crystal structure of M. leprae ML2640c defines a large family of putative S-adenosylmethionine-dependent methyltransferases in mycobacteria

Mycobacterium leprae protein ML2640c belongs to a large family of conserved hypothetical proteins predominantly found in mycobacteria, some of them predicted as putative S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTase). As part of a Structural Genomics initiative on conserved hypothetical proteins in pathogenic mycobacteria, we have determined the structure of ML2640c in two distinct crystal forms. As expected, ML2640c has a typical MTase core domain and binds the methyl donor substrate AdoMet in a manner consistent with other known members of this structural family. The putative acceptor substrate-binding site of ML2640c is a large internal cavity, mostly lined by aromatic and aliphatic side-chain residues, suggesting that a lipid-like molecule might be targeted for catalysis. A flap segment (residues 222-256), which isolates the binding site from the bulk solvent and is highly mobile in the crystal structures, could serve as a gateway to allow substrate entry and product release. The multiple sequence alignment of ML2640c-like proteins revealed that the central alpha/beta core and the AdoMet-binding site are very well conserved within the family. However, the amino acid positions defining the binding site for the acceptor substrate display a higher variability, suggestive of distinct acceptor substrate specificities. The ML2640c crystal structures offer the first structural glimpses at this important family of mycobacterial proteins and lend strong support to their functional assignment as AdoMet-dependent methyltransferases.     

Molecular mechanism underlying the interaction of typical Sac10b family proteins with DNA

The Sac10b protein family is regarded as a family of DNA-binding proteins that is highly conserved and widely distributed within the archaea. Sac10b family members are typically small basic dimeric proteins that bind to DNA with cooperativity and no sequence specificity and are capable of constraining DNA negative supercoils, protecting DNA from Dnase I digestion, and do not compact DNA obviously. However, a detailed understanding of the structural basis of the interaction of Sac10b family proteins with DNA is still lacking. Here, we determined the crystal structure of Mth10b, an atypical member of the Sac10b family from Methanobacterium thermoautotrophicum ΔH, at 2.2 Å. Unlike typical Sac10b family proteins, Mth10b is an acidic protein and binds to neither DNA nor RNA. The overall structure of Mth10b displays high similarity to its homologs, but three pairs of conserved positively charged residues located at the presumed DNA-binding surface are substituted by non-charged residues in Mth10b. Through amino acids interchanges, the DNA-binding ability of Mth10b was restored successfully, whereas the DNA-binding ability of Sso10b, a typical Sac10b family member, was weakened greatly. Based on these results, we propose a model describing the molecular mechanism underlying the interactions of typical Sac10b family proteins with DNA that explains all the characteristics of the interactions between typical Sac10b family members and DNA.     

Crystal structure of a putative CN hydrolase from yeast

The crystal structure of a yeast hypothetical protein with sequence similarity to CN hydrolases has been determined to 2.4 A resolution by the multiwavelength anomalous dispersion (MAD) method. The protein folds as a four-layer alphabetabetaalpha sandwich and exists as a dimer in the crystal and in solution. It was selected in a structural genomics project as representative of CN hydrolases at a time when no structures had been determined for members of this family. Structures for two other members of the family have since been reported and the three proteins have similar topology and dimerization modes, which are distinct from those of other alphabetabetaalpha proteins whose structures are known. The dimers form an unusual eight-layer alphabetabetaalpha:alphabetabetaalpha structure. Although the precise enzymatic reactions catalyzed by the yeast protein are not known, considerable information about the active site may be deduced from conserved sequence motifs, comparative biochemical information, and comparison with known structures of hydrolase active sites. As with serine hydrolases, the active-site nucleophile (cysteine in this case) is positioned on a nucleophile elbow.     

The structure of the outer membrane protein OmpX from Escherichia coli reveals possible mechanisms of virulence.

BACKGROUND: The integral outer membrane protein X (OmpX) from Escherichia coli belongs to a family of highly conserved bacterial proteins that promote bacterial adhesion to and entry into mammalian cells. Moreover, these proteins have a role in the resistance against attack by the human complement system. Here we present the first crystal structure of a member of this family. RESULTS: The crystal structure of OmpX from E. coli was determined at 1.9 A resolution using multiple isomorphous replacement. OmpX consists of an eight-stranded antiparallel all-next-neighbor beta barrel. The structure shows two girdles of aromatic amino acid residues and a ribbon of nonpolar residues that attach to the membrane interior. The core of the barrel consists of an extended hydrogen-bonding network of highly conserved residues. OmpX thus resembles an inverse micelle. The structure explains the dramatically improved crystal quality of OmpX containing the mutation His100-->Asn, which made the X-ray analysis possible. The coordination spheres of two bound platinum ions are described. CONCLUSIONS: The OmpX structure shows that within a family of virulence-related membrane proteins, the membrane-spanning part of the protein is much better conserved than the extracellular loops. Moreover, these loops form a protruding beta sheet, the edge of which presumably binds to external proteins. It is suggested that this type of binding promotes cell adhesion and invasion and helps defend against the complement system. Although OmpX has the same beta-sheet topology as the structurally related outer membrane protein A (OmpA), their barrels differ with respect to the shear numbers and internal hydrogen-bonding networks.     

Malarial EBA-175 region VI crystallographic structure reveals a KIX-like binding interface

The malaria parasite proliferates in the bloodstream of its vertebrate host by invading and replicating within erythrocytes. To achieve successful invasion, a number of discrete and essential events need to take place at the parasite-host cell interface. Erythrocyte-binding antigen 175 (EBA-175) is a member of a family of Plasmodium falciparum erythrocyte-binding proteins involved in the formation of a tight junction, a necessary step in invasion. Here we present the crystal structure of EBA-175 region VI (rVI), a cysteine-rich domain that is highly conserved within the protein family and is essential for EBA-175 trafficking. The structure was solved by selenomethionine single-wavelength anomalous dispersion at 1.8 Å resolution. It reveals a homodimer, containing in each subunit a compact five-α-helix core that is stabilized by four conserved disulfide bridges. rVI adopts a novel fold that is likely conserved across the protein family, indicating a conserved function. It shows no similarity to the Duffy-binding-like domains of EBA-175 involved in erythrocyte binding, indicating a distinct role. Remarkably, rVI possesses structural features related to the KIX-binding domain of the coactivator CREB-binding protein, supporting the binding and trafficking roles that have been ascribed to it and providing a rational basis for further experimental investigation of its function.     

Crystal structure of YfeU protein from Haemophilus influenzae: a predicted etherase involved in peptidoglycan recycling

The crystal structure of the H. influenzae YfeU protein, was determined at 1.90 Å resolution using multi-wavelength anomalous diffraction. YfeU belongs to a very large conserved family of proteins found mainly in bacteria but also in archaea and eukaryota. The protein is a homolog of eukaryotic glucokinase regulator and is predicted to be a sugar phosphate isomerase or aminotransferase. Here we describe the structure of YfeU and discuss the possible function as an etherase possibly involved in peptidoglycan recycling.     

1.2 Angstroms crystal structure of the S. pneumoniae PhtA histidine triad domain a novel zinc binding fold

The recently described pneumococcal histidine triad protein family has been shown to be highly conserved within the pneumococcus. As part of our structural genomics effort on proteins from Streptococcus pneumoniae, we have expressed, crystallised and solved the structure of PhtA-166-220 at 1.2 Angstroms using remote SAD with zinc. The structure of PhtA-166-220 shows no similarity to any protein structure. The overall fold contains 3beta-strands and a single short alpha-helix. The structure appears to contain a novel zinc binding motif. The remaining 4 histidine triad repeats from PhtA have been modelled based on the crystal structure of the PhtA histidine triad repeat 2. From this modelling work, we speculate that only three of the five histidine triad repeats contain the residues in the correct geometry to allow the binding of a zinc ion.     

Crystal structure of a dimerization domain of human Caprin-2: similar overall dimeric fold but different molecular surface properties to that of human Caprin-1

Abstract

Human Caprin-1 and Caprin-2 are prototypic members of the caprin (cytoplasmic activation/proliferation-associated protein) protein family. Vertebrate caprin proteins contain two highly conserved homologous regions (HR1 and HR2) and C-terminal RGG motifs. Drosophila caprin (dCaprin) shares HR1 and RGG motifs but lacks HR2. Caprin-1 and Caprin-2 have important and non-redundant functions. The detailed molecular mechanisms of their actions remain largely unknown. Previously, we determined the crystal structure of a ～120-residue fragment of Caprin-1 within the HR1 region. The structure has a novel all α-helical fold that self-associates to form a homodimer. In this study, the crystal structure of a corresponding fragment from Caprin-2 is reported. The Caprin-2 fragment has similar protein fold and dimeric structure as that of the Caprin-1 fragment. Structural comparison reveals that the molecular interactions mediating homodimerization of Caprin-1 and Caprin-2 are largely conserved in the two systems. Structural-modelling study of the corresponding dCaprin fragment indicates that dCaprin may also adopt a similar dimeric structure. The presence of a dimerization domain within HR1 may represent an evolutionarily conserved feature of the caprin protein family. Interestingly, while Caprin-1 and Caprin-2 adopt similar overall dimeric structures, the two structures have quite different molecular surface properties. In the Caprin-1 dimeric structure, some of the surface areas are known or suspected to function as binding sites for Carpin-1-interacting proteins. The different surface properties of the caprin dimeric structures may dictate their intermolecular interaction with specific protein partners.

Communicated by Ramaswamy H. Sarma.     

Selective recognition of mannose by the human eosinophil Charcot-Leyden crystal protein (galectin-10): a crystallographic study at 1.8 A resolution.

The role(s) of the eosinophil Charcot-Leyden crystal (CLC) protein in eosinophil or basophil function or associated inflammatory processes is yet to be established. Although the CLC protein has been reported to exhibit weak lysophospholipase activity, it shows virtually no sequence homology to any known member of this family of enzymes. The X-ray crystal structure of the CLC protein is very similar to the structure of the galectins, members of a beta-galactoside-specific animal lectin family, including a partially conserved galectin carbohydrate recognition domain (CRD). In the absence of any known natural carbohydrate ligand for this protein, the functional role of the CLC protein (galectin-10) has remained speculative. Here we describe structural studies on the carbohydrate binding properties of the CLC protein and report the first structure of a carbohydrate in complex with the protein. Interestingly, the CLC protein demonstrates no affinity for beta-galactosides and binds mannose in a manner very different from those of other related galectins that have been shown to bind lactosamine. The partial conservation of residues involved in carbohydrate binding led to significant changes in the topology and chemical nature of the CRD, and has implications for carbohydrate recognition by the CLC protein in vivo and its functional role in the biology of inflammation.     

The first structure of a glycoside hydrolase family 61 member, Cel61B from Hypocrea jecorina, at 1.6 A resolution

The glycoside hydrolase (GH) family 61 is a long-recognized, but still recondite, class of proteins, with little known about the activity, mechanism or function of its more than 70 members. The best-studied GH family 61 member, Cel61A of the filamentous fungus Hypocrea jecorina, is known to be an endoglucanase, but it is not clear if this represents the main activity or function of this family in vivo. We present here the first structure for this family, that of Cel61B from H. jecorina. The best-quality crystals were formed in the presence of nickel, and the crystal structure was solved to 1.6 Å resolution using a single-wavelength anomalous dispersion method with nickel as the source of anomalous scatter. Cel61B lacks a carbohydrate-binding module and is a single-domain protein that folds into a twisted β-sandwich. A structure-aided sequence alignment of all GH family 61 proteins identified a highly conserved group of residues on the surface of Cel61B. Within this patch of mostly polar amino acids was a site occupied by the intramolecular nickel hexacoordinately bound in the solved structure. In the Cel61B structure, there is no easily identifiable carbohydrate-binding cleft or pocket or catalytic center of the types normally seen in GHs. A structural comparison search showed that the known structure most similar to Cel61B is that of CBP21 from the Gram-negative soil bacterium Serratia marcescens, a member of the carbohydrate-binding module family 33 proteins. A polar surface patch highly conserved in that structural family has been identified in CBP21 and shown to be involved in chitin binding and in the protein's enhancement of chitinase activities. The analysis of the Cel61B structure is discussed in light of our continuing research to better understand the activities and function of GH family 61.     

Crystal structure of the YgfZ protein from Escherichia coli suggests a folate-dependent regulatory role in one-carbon metabolism

The ygfZ gene product of Escherichia coli represents a large protein family conserved in bacteria to eukaryotes. The members of this family are uncharacterized proteins with marginal sequence similarity to the T-protein (aminomethyltransferase) of the glycine cleavage system. To assist with the functional assignment of the YgfZ family, the crystal structure of the E. coli protein was determined by multiwavelength anomalous diffraction. The protein molecule has a three-domain architecture with a central hydrophobic channel. The structure is very similar to that of bacterial dimethylglycine oxidase, an enzyme of the glycine betaine pathway and a homolog of the T-protein. Based on structural superposition, a folate-binding site was identified in the central channel of YgfZ, and the ability of YgfZ to bind folate derivatives was confirmed experimentally. However, in contrast to dimethylglycine oxidase and T-protein, the YgfZ family lacks amino acid conservation at the folate site, which implies that YgfZ is not an aminomethyltransferase but is likely a folate-dependent regulatory protein involved in one-carbon metabolism.     

Crystal structure of the core domain of RhoE/Rnd3: a constitutively activated small G protein

We report the 2.1 A crystal structure of the core G protein domain of the unusual Rho family member RhoE/Rnd3 in complex with endogenous GTP and magnesium. Unlike other small G proteins, RhoE, along with two other proteins Rnd1/Rho6 and Rnd2/RhoN, does not hydrolyze GTP. The main reason for this is the presence of serines in the positions equivalent to Ala59 and Gln61 in Ras. The structure shows that there are still water molecules in similar positions to the waters thought to be involved in the hydrolysis reaction in other G proteins. The structure suggests three not necessarily exclusive explanations for the lack of hydrolysis. The lack of the conserved glutamine raises the energy of the transition state inhibiting hydrolysis. The serines may restrain the waters from moving closer to the GTP, a step that is required to attain the transition state. They also stabilize the GTP-bound conformation of switch II and could prevent conformational changes required during hydrolysis. By superposition of the RhoE structure on structures of Rho family proteins in complex with binding partners, we make predictions on RhoE interactions with these partners.     

Crystal structure of the GAP domain of Gyp1p: first insights into interaction with Ypt/Rab proteins

We present the 1.9 A resolution crystal structure of the catalytic domain of Gyp1p, a specific GTPase activating protein (GAP) for Ypt proteins, the yeast homologues of Rab proteins, which are involved in vesicular transport. Gyp1p is a member of a large family of eukaryotic proteins with shared sequence motifs. Previously, no structural information was available for any member of this class of proteins. The GAP domain of Gyp1p was found to be fully alpha-helical. However, the observed fold does not superimpose with other alpha-helical GAPs (e.g. Ras- and Cdc42/Rho-GAP). The conserved and catalytically crucial arginine residue, identified by mutational analysis, is in a comparable position to the arginine finger in the Ras- and Cdc42-GAPs, suggesting that Gyp1p utilizes an arginine finger in the GAP reaction, in analogy to Ras- and Cdc42-GAPs. A model for the interaction between Gyp1p and the Ypt protein satisfying biochemical data is given.     

Crystal structure of a 123 amino acids dimerization domain of Drosophila Caprin

Cytoplasmic activation/proliferation-associated protein (Caprin) proteins assume diverse functions in many important biological processes, including synaptic plasticity, stress response, innate immune response, and cellular proliferation. The Caprin family members are characterized by the presence of a highly conserved homologous region (HR1) at the N-terminus and arginine-glycine-rich (RGG) boxes at the C-terminus. We had previously determined the crystal structures of human Caprin-1 and Caprin-2 fragments corresponding to the C-terminal 2/3 of HR1. Both fragments adopt homodimeric structures. Based on sequence conservation, we speculated that all Caprin proteins should have similar homodimeric structures. Here we report the crystal structure of a fragment (residues 187-309) of Drosophila melanogaster Caprin (dCaprin). The dCaprin fragment adopts an all α-helical fold which self-associates to form a homodimer. The overall dCaprin homodimeric structure is similar to the Caprin-1 and Caprin-2 homodimeric structures. Most of the amino acids residues mediating homodimerization in the three structures are conserved among all Caprin family members. These structural and sequence data suggest that homodimerization through a conserved dimerization domain is a common structural feature of the Caprin protein family. The dimeric structures may also be involved in interaction with Caprin partners. Dimer formation creates a V-shape concave surface that may serve as a protein binding groove. The concave surfaces in Caprin-1, Caprin-2, and dCaprin should have different and specific binding partners due to the large difference in electrostatic potentials. We propose the existence of a multi-functional domain in Caprin proteins, which not only mediate homodimerization but also involve in interaction with specific Caprin partners.     

Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif-like domain

We have determined the crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 at 1.9 A resolution. This protein is a member of the Escherichia coli ygcH sequence family, which contains approximately 15 sequence homologs of bacterial origin. These homologs have a high isoelectric point. The crystal structure reveals that TTHB192 consists of two independently folded domains, and that each domain exhibits a ferredoxin-like fold with a four-stranded antiparallel beta-sheet packed on one side by alpha-helices. These two tandem domains face each other to generate a beta-sheet platform. TTHB192 displays overall structural similarity to Sex-lethal protein and poly(A)-binding protein fragments. These proteins have RNA binding activity which is supported by a beta-sheet platform formed by two tandem repeats of an RNA recognition motif domain with signature sequence motifs on the beta-sheet surface. Although TTHB192 does not have the same signature sequence motif as the RNA recognition motif domain, the presence of an evolutionarily conserved basic patch on the beta-sheet platform could be functionally relevant for nucleic acid-binding. This report shows that TTHB192 and its sequence homologs adopt an RNA recognition motif-like domain and provides the first testable functional hypothesis for this protein family.     

Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins

The RecA family of proteins mediates homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against DNA double strand breaks. RecA proteins are thought to facilitate DNA strand exchange reactions as closed-rings or as right-handed helical filaments. Here, we report the crystal structure of a left-handed Sulfolobus solfataricus RadA helical filament. Each protomer in this left-handed filament is linked to its neighbour via interactions of a β-strand polymerization motif with the neighbouring ATPase domain. Immediately following the polymerization motif, we identified an evolutionarily conserved hinge region (a subunit rotation motif) in which a 360° clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the AMP–PNP right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. Additional structural and functional analyses of wild-type and mutant proteins confirmed that the subunit rotation motif is crucial for enzymatic functions of RecA family proteins. These observations support the hypothesis that RecA family protein filaments may function as rotary motors.