Dimer Interface Migration in a Viral Sulfhydryl Oxidase

Large double-stranded DNA viruses, including poxviruses and mimiviruses, encode enzymes to catalyze the formation of disulfide bonds in viral proteins produced in the cell cytosol, an atypical location for oxidative protein folding. These viral disulfide catalysts belong to a family of sulfhydryl oxidases that are dimers of a small five-helix fold containing a Cys-X-X-Cys motif juxtaposed to a flavin adenine dinucleotide cofactor. We report that the sulfhydryl oxidase pB119L from African swine fever virus (ASFV) uses for self-assembly surface different from that observed in homologs from mammals, plants, and fungi. Within a protein family, different packing interfaces for the same oligomerization state are extremely rare. We find that the alternate dimerization mode seen in ASFV pB119L is not characteristic of all viral sulfhydryl oxidases, as the flavin-binding domain from a mimivirus sulfhydryl oxidase assumes the same dimer structure as the known eukaryotic enzymes. ASFV pB119L demonstrates the potential of large double-stranded DNA viruses, which have faster mutation rates than their hosts and the tendency to incorporate host genes, to pioneer new protein folds and self-assembly modes.     

Analysis of interspecies variability of immunoglobulin lambda chains reveals differences in domain tertiary structures.

X-ray crystallography showed the NH₂-proximal (variable, V) and COOH-proximal (constant, C) halves of the λ chain to fold independently into autonomous three-dimensional domains, homologous domains from different chains interacting strongly by non-covalent forces to form domain dimers. In this letter, interspecies homology of five immunoglobulin λ chain primary structures from man, mouse and pig has been evaluated separately for those segments of polypeptide chains that correspond to basic structural elements of a domain fold. The highest degree of phylogenetic conservation has always been found with segments forming the innermost part of domain dimers, though the V-V dimer is known to be packed inside out with respect to the C-C dimer, its inner segments being different from those of the C-C dimer. Differences of interspecies homology between analogous segments from the variable and the constant domain have been revealed which correlate with differences in domain architectures. Two different measures of interspecies homology have been defined (% positions with identical residues; % positions displaying 80% homology) the ratio of which is indicative of packing density and of folding constraints imposed on a particular segment.     

Structural basis for antifreeze activity of ice-binding protein from arctic yeast

Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ～25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-? resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96-115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region ((243)PFVPAPEVV(251)). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn(185) provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins.     

Identification, characterization, and crystal structure of Bacillus subtilis nicotinic acid mononucleotide adenylyltransferase.

The nadD gene, encoding the enzyme nicotinic acid mononucleotide (NaMN) adenylyltransferase (AT), is essential for the synthesis of NAD and subsequent viability of the cell. The nadD gene in Bacillus subtilis (yqeJ) was identified by sequence homology with other bacterial nadD genes and by biochemical characterization of the gene product. NaMN AT catalyzes the reversible adenylation of both NaMN and the nicotinamide mononucleotide (NMN) but shows specificity for the nicotinate. In contrast to other known NMN ATs, biophysical characterizations reveal it to be a dimer. The NaMN AT crystal structure was determined for both the apo enzyme and product-bound form, to 2.1 and 3.2 A, respectively. The structures reveal a "functional" dimer conserved in both crystal forms and a monomer fold common to members of the nucleotidyl-transferase alpha/beta phosphodiesterase superfamily. A structural comparison with family members suggests a new conserved motif (SXXXX(R/K)) at the N terminus of an alpha-helix, which is not part of the shared fold. Interactions of the nicotinic acid with backbone atoms indicate the structural basis for specificity.     

Propensity for C-terminal domain swapping correlates with increased regional flexibility in the C-terminus of RNase A

Domain swapping is a type of oligomerization in which monomeric proteins exchange a structural element, resulting in oligomers whose subunits recapitulate the native, monomeric fold. It has been implicated as a potential mechanism for protein aggregation, which provides a strong impetus to understand the structural determinants and folding mechanisms that trigger domain swapping. Bovine pancreatic ribonuclease A (RNase A) is a well-studied protein known to domain swap under extreme conditions, such as lyophilization from acetic acid. The major domain-swapped dimer form of RNase A exchanges a β-strand at its C-terminus to form a C-terminal domain-swapped dimer. To study the mechanism by which C-terminal swapping occurs, we used a variant of RNase A containing a P114G mutation that readily domain swaps under physiological conditions. Using NMR and hydrogen-deuterium exchange, we find that the P114G variant has decreased protection from hydrogen exchange compared to the wild-type protein near the C-terminal hinge region. Our results suggest that domain swapping occurs via a local high-energy fluctuation at the C-terminus.     

Characterization of mimivirus DNA topoisomerase IB suggests horizontal gene transfer between eukaryal viruses and bacteria

Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes dozens of proteins with imputed functions in nucleic acid transactions. Here we produced, purified, and characterized mimivirus DNA topoisomerase IB (TopIB), which we find to be a structural and functional homolog of poxvirus TopIB and the poxvirus-like topoisomerases discovered recently in bacteria. Arginine, histidine, and tyrosine side chains responsible for TopIB transesterification are conserved and essential in mimivirus TopIB. Moreover, mimivirus TopIB is capable of incising duplex DNA at the 5'-CCCTT cleavage site recognized by all poxvirus topoisomerases. Based on the available data, mimivirus TopIB appears functionally more akin to poxvirus TopIB than bacterial TopIB, despite its greater primary structure similarity to the bacterial TopIB group. We speculate that the ancestral bacterial/viral TopIB was disseminated by horizontal gene transfer within amoebae, which are permissive hosts for either intracellular growth or persistence of many present-day bacterial species that have a type IB topoisomerase.     

Mammalian SCAN domain dimer is a domain-swapped homolog of the HIV capsid C-terminal domain

Retroviral assembly is driven by multiple interactions mediated by the Gag polyprotein, the main structural component of the forming viral shell. Critical determinants of Gag oligomerization are contained within the C-terminal domain (CTD) of the capsid protein, which also harbors a conserved sequence motif, the major homology region (MHR), in the otherwise highly variable Gag. An unexpected clue about the MHR function in retroviral assembly emerges from the structure of the zinc finger-associated SCAN domain we describe here. The SCAN dimer adopts a fold almost identical to that of the retroviral capsid CTD but uses an entirely different dimerization interface caused by swapping the MHR-like element between the monomers. Mutations in retroviral capsid proteins and functional data suggest that a SCAN-like MHR-swapped CTD dimer forms during immature particle assembly. In the SCAN-like dimer, the MHR contributes the major part of the large intertwined dimer interface explaining its functional significance.     

The N-terminal cysteine pair of yeast sulfhydryl oxidase Erv1p is essential for in vivo activity and interacts with the primary redox centre.

Yeast Erv1p is a ubiquitous FAD-dependent sulfhydryl oxidase, located in the intermembrane space of mitochondria. The dimeric enzyme is essential for survival of the cell. Besides the redox-active CXXC motif close to the FAD, Erv1p harbours two additional cysteine pairs. Site-directed mutagenesis has identified all three cysteine pairs as essential for normal function. The C-terminal cysteine pair is of structural importance as it contributes to the correct arrangement of the FAD-binding fold. Variations in dimer formation and unique colour changes of mutant proteins argue in favour of an interaction between the N-terminal cysteine pair with the redox centre of the partner monomer.     

An Src homology 3-like domain is responsible for dimerization of the repressor protein KorB encoded by the promiscuous IncP plasmid RP4.

KorB is a regulatory protein encoded by the conjugative plasmid RP4 and a member of the ParB family of bacterial partitioning proteins. The protein regulates the expression of plasmid genes whose products are involved in replication, transfer, and stable inheritance of RP4 by binding to palindromic 13-bp DNA sequences (5'-TTTAGC(G/C)GCTAAA-3') present 12 times in the 60-kb plasmid. Here we report the crystal structure of KorB-C, the C-terminal domain of KorB comprising residues 297-358. The structure of KorB-C was solved in two crystal forms. Quite unexpectedly, we find that KorB-C shows a fold closely resembling the Src homology 3 (SH3) domain, a fold well known from proteins involved in eukaryotic signal transduction. From the arrangement of molecules in the asymmetric unit, it is concluded that two molecules form a functionally relevant dimer. The detailed analysis of the dimer interface and a chemical cross-linking study suggest that the C-terminal domain is responsible for stabilizing the dimeric form of KorB in solution to facilitate binding to the palindromic operator sequence. The KorB-C crystal structure extends the range of protein-protein interactions known to be promoted by SH3 and SH3-like domains.     

Crystal structure of receiver domain of putative NarL family response regulator spr1814 from Streptococcus pneumoniae in the absence and presence of the phosphoryl analog beryllofluoride

Spr1814 of Streptococcus pneumoniae is a putative response regulator (RR) that has four-helix helix-turn-helix DNA-binding domain and belongs to the NarL family. The prototypical RR contains two domains, an N-terminal receiver domain linked to a variable effector domain. The receiver domain functions as a phosphorylation-activated switch and contains the typical doubly wound five-stranded α/β fold. Here, we report the crystal structure of the receiver domain of spr1814 (spr1814(R)) determined in the absence and presence of beryllofluoride as a phosphoryl analog. Based on the overall structure, spr1814(R) was shown to contain the typical fold similar with other structures of the receiver domain; however, an additional linker region connecting the receiver and DNA-binding domain was inserted into the dimer interface of spr1814(R), resulting in the formation of unique dimer interface. Upon phosphorylation, the conformational change of the linker region was observed and this suggests that domain rearrangement between the receiver domain and effector domain could occur in full-length spr1814.     

Structure of the parathyroid hormone receptor C terminus bound to the G-protein dimer Gbeta1gamma2

A critical role of the Gbetagamma dimer in heterotrimeric G-protein signaling is to facilitate the engagement and activation of the Galpha subunit by cell-surface G-protein-coupled receptors. However, high-resolution structural information of the connectivity between receptor and the Gbetagamma dimer has not previously been available. Here, we describe the structural determinants of Gbeta1gamma2 in complex with a C-terminal region of the parathyroid hormone receptor-1 (PTH1R) as obtained by X-ray crystallography. The structure reveals that several critical residues within PTH1R contact only Gbeta residues located within the outer edge of WD1- and WD7-repeat segments of the Gbeta toroid structure. These regions encompass a predicted membrane-facing region of Gbeta thought to be oriented in a fashion that is accessible to the membrane-spanning receptor. Mutation of key receptor contact residues on Gbeta1 leads to a selective loss of function in receptor/heterotrimer coupling while preserving Gbeta1gamma2 activation of the effector phospholipase-C beta.     

Domain organization of Salmonella typhimurium formylglycinamide ribonucleotide amidotransferase revealed by X-ray crystallography

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent conversion of formylglycinamide ribonucleotide (FGAR) and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, P(i), and glutamate in the fourth step of the purine biosynthetic pathway. In eukaryotes and Gram-negative bacteria, FGAR-AT is encoded by the purL gene as a multidomain protein with a molecular mass of about 140 kDa. In Gram-positive bacteria and archaebacteria FGAR-AT is a complex of three proteins: PurS, PurL, and PurQ. We have determined the structure of FGAR-AT (PurL) from Salmonella typhimurium at 1.9 A resolution using X-ray crystallography. PurL is the last remaining enzyme in the purine biosynthetic pathway to have its structure determined. The structure reveals four domains: an N-terminal domain structurally homologous to a PurS dimer, a linker region, an FGAM synthetase domain homologous to an aminoimidazole ribonucleotide synthetase (PurM) dimer, and a triad glutaminase domain. The domains are intricately linked by interdomain interactions and peptide connectors. The fold common to PurM and the central region of PurL represents a superfamily for which HypE, SelD, and ThiL are predicted to be members. A structural ADP molecule was found bound to a site related to the putative active site by pseudo-2-fold symmetry and two sulfate ions were found at the putative active site. These observations and the structural similarities between PurM and StPurL were used to model the substrates FGAR and ATP in the StPurL active site. A glutamylthioester intermediate was found in the glutaminase domain at Cys1135. The N-terminal (PurS-like) domain is hypothesized to form the putative channel through which ammonia passes from the glutaminase domain to the FGAM synthetase domain.     

Structural features and the persistence of acquired proteins

ORFan genes can constitute a large fraction of a bacterial genome, but due to their lack of homologs, their functions have remained largely unexplored. To determine if particular features of ORFan-encoded proteins promote their presence in a genome, we analyzed properties of ORFans that originated over a broad evolutionary timescale. We also compared ORFan genes to another class of acquired genes, heterogeneous occurrence in prokaryotes (HOPs), which have homologs in other bacteria. A total of 54 ORFan and HOP genes selected from different phylogenetic depths in the Escherichia coli lineage were cloned, expressed, purified, and subjected to circular dichroism (CD) spectroscopy. A majority of genes could be expressed, but only 18 yielded sufficient soluble protein for spectral analysis. Of these, half were significantly alpha-helical, three were predominantly beta-sheet, and six were of intermediate/indeterminate structure. Although a higher proportion of HOPs yielded soluble proteins with resolvable secondary structures, ORFans resembled HOPs with regard to most of the other features tested. Overall, we found that those ORFan and HOP genes that have persisted in the E. coli lineage were more likely to encode soluble and folded proteins, more likely to display environmental modulation of their gene expression, and by extrapolation, are more likely to be functional.     

A structural model for the rolA protein and its interaction with DNA

The study of the plant oncogene rolA has been hampered by a lack of structural information. Here we show that, despite a lack of significant sequence similarity to proteins of known structure, the rolA sequence adopts a known fold; that of the papillomavirus E2 DNA-binding domain. This fold is reliably identified by modern threading programs, which consider predicted secondary structure, but not by others. Although the rolA sequence is only around 16% identical to those of the available template structures, a structural model could be built that performed well against protein structure verification programs. The adopted strategy involved alignment corrections, justified by multiple model building and evaluation, with particular attention paid to the hydrophobic core residues. We find that rolA protein is predicted to resemble the template proteins in two key aspects; existence as a dimer and ability to bind DNA. rolA protein has recently been shown experimentally to possess DNA binding ability. This model predicts Lys 24 and Arg 27 to be involved in sequence-specific interactions and eight other residues to hydrogen-bond phosphate groups of the DNA.     

Dimer dissociation is a key energetic event in the fold-switch pathway of KaiB

Cyanobacteria possesses the simplest circadian clock, composed of three proteins that act as a phosphorylation oscillator: KaiA, KaiB, and KaiC. The timing of this oscillator is determined by the fold-switch of KaiB, a structural rearrangement of its C-terminal half that is accompanied by a change in the oligomerization state. During the day, KaiB forms a stable tetramer (gsKaiB), whereas it adopts a monomeric thioredoxin-like fold during the night (fsKaiB). Although the structures and functions of both native states are well studied, little is known about the sequence and structure determinants that control their structural interconversion. Here, we used confinement molecular dynamics (CCR-MD) and folding simulations using structure-based models to show that the dissociation of the gsKaiB dimer is a key energetic event for the fold-switch. Hydrogen-deuterium exchange mass spectrometry (HDXMS) recapitulates the local stability of protein regions reported by CCR-MD, with both approaches consistently indicating that the energy and backbone flexibility changes are solely associated with the region that fold-switches between gsKaiB and fsKaiB and that the localized regions that differentially stabilize gsKaiB also involve regions outside the dimer interface. Moreover, two mutants (R23C and R75C) previously reported to be relevant for altering the rhythmicity of the Kai clock were also studied by HDXMS. Particularly, R75C populates dimeric and monomeric states with a deuterium incorporation profile comparable to the one observed for fsKaiB, emphasizing the importance of the oligomerization state of KaiB for the fold-switch. These findings suggest that the information necessary to control the rhythmicity of the cyanobacterial biological clock is, to a great extent, encoded within the KaiB sequence.     

Processing of cholinesterase-like α/β-hydrolase fold proteins: alterations associated with congenital disorders

The α/β hydrolase fold family is perhaps the largest group of proteins presenting significant structural homology with divergent functions, ranging from catalytic hydrolysis to heterophilic cell adhesive interactions to chaperones in hormone production. All the proteins of the family share a common three-dimensional core structure containing the α/β hydrolase fold domain that is crucial for proper protein function. Several mutations associated with congenital diseases or disorders have been reported in conserved residues within the α/β-hydrolase fold domain of cholinesterase-like proteins, neuroligins, butyrylcholinesterase and thyroglobulin. These mutations are known to disrupt the architecture of the common structural domain either globally or locally. Characterization of the natural mutations affecting the α/β-hydrolase fold domain in these proteins has shown that they mainly impair processing and trafficking along the secretory pathway causing retention of the mutant protein in the endoplasmic reticulum. Studying the processing of α/β-hydrolase fold mutant proteins should uncover new functions for this domain, that in some cases require structural integrity for both export of the protein from the ER and for facilitating subunit dimerization. A comparative study of homologous mutations in proteins that are closely related family members, along with the definition of new three-dimensional crystal structures, will identify critical residues for the assembly of the α/β-hydrolase fold.     

Structure of a novel Bence-Jones protein (Rhe) fragment at 1.6 A resolution

The crystal structure of Rhe, a lambda-type Bence-Jones protein fragment, has been solved and refined to a resolution of 1.6 A. A model fragment consisting of the complete variable domain and the first three residues of the constant domain yields a crystallographic residual RF value of 0.149. The protein exists as a dimer both in solution and in the crystals. Although the "immunoglobulin fold" is generally preserved in the structure, there are significant differences in both the monomer conformation and in the mode of association of monomers into dimers, when compared to other known Bence-Jones proteins or Fab fragments. The variations in conformation within monomers are particularly significant as they involve non-hypervariable residues, which previously were believed to be part of a "structurally invariant" framework common to all immunoglobulin variable domains. The novel mode of dimerization is equally important, as it can result in combining site shapes and sizes unobtainable with the conventional mode of dimerization. A comparison of the structure with other variable domain dimers reveals further that the variations within monomers and between domains in the dimer are coupled. Some possible functional implications revealed by this coupling are greater variability, induced fitting of the combining site to better accommodate antigenic determinants, and a mechanism for relaying binding information from one end of the variable domain dimer to the other. In addition to providing the most accurate atomic parameters for an immunoglobulin domain yet obtained, the high resolution and extensive refinement resulted in identification of several tightly bound water molecules in key structural positions. These water molecules may be regarded as integral components of the protein. Other water molecules appear to be required to stabilize the novel conformation.