A novel member of the split betaalphabeta fold: Solution structure of the hypothetical protein YML108W from Saccharomyces cerevisiae

As part of the Northeast Structural Genomics Consortium pilot project focused on small eukaryotic proteins and protein domains, we have determined the NMR structure of the protein encoded by ORF YML108W from Saccharomyces cerevisiae. YML108W belongs to one of the numerous structural proteomics targets whose biological function is unknown. Moreover, this protein does not have sequence similarity to any other protein. The NMR structure of YML108W consists of a four-stranded beta-sheet with strand order 2143 and two alpha-helices, with an overall topology of betabetaalphabetabetaalpha. Strand beta1 runs parallel to beta4, and beta2:beta1 and beta4:beta3 pairs are arranged in an antiparallel fashion. Although this fold belongs to the split betaalphabeta family, it appears to be unique among this family; it is a novel arrangement of secondary structure, thereby expanding the universe of protein folds.     

Solution NMR structure of Alr2454 from Nostoc sp. PCC 7120, the first structural representative of Pfam domain family PF11267

Protein domain family PF11267 (DUF3067) is a family of proteins of unknown function found in both bacteria and eukaryotes. Here we present the solution NMR structure of the 102-residue Alr2454 protein from Nostoc sp. PCC 7120, which constitutes the first structural representative from this conserved protein domain family. The structure of Nostoc sp. Alr2454 adopts a novel protein fold.     

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.     

Three structural representatives of the PF06855 protein domain family from Staphyloccocus aureus and Bacillus subtilis have SAM domain-like folds and different functions

Protein domain family PF06855 (DUF1250) is a family of small domains of unknown function found only in bacteria, and mostly in the order Bacillales and Lactobacillales. Here we describe the solution NMR or X-ray crystal structures of three representatives of this domain family, MW0776 and MW1311 from Staphyloccocus aureus and yozE from Bacillus subtilis. All three proteins adopt a four-helix motif similar to sterile alpha motif (SAM) domains. Phylogenetic analysis classifies MW1311 and yozE as functionally equivalent proteins of the UPF0346 family of unknown function, but excludes MW0776, which likely has a different biological function. Our structural characterization of the three domains supports this separation of function. The structures of MW0776, MW1311, and yozE constitute the first structural representatives from this protein domain family.     

Solution structure of the single-stranded DNA binding protein of the filamentous Pseudomonas phage Pf3: similarity to other proteins binding to single-stranded nucleic acids.

The three-dimensional structure of the homodimeric single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been determined using heteronuclear multidimensional NMR techniques and restrained molecular dynamics. NMR experiments and structure calculations have been performed on a mutant protein (Phe36 --> His) that was successfully designed to reduce the tendency of the protein to aggregate. The protein monomer is composed of a five-stranded antiparallel beta-sheet from which two beta-hairpins and a large loop protrude. The structure is compared with the single-stranded DNA binding protein encoded by the filamentous Escherichia coli phage Ff, a protein with a similar biological function and DNA binding properties, yet quite different amino acid sequence, and with the major cold shock protein of Escherichia coli, a single-stranded DNA binding protein with an entirely different sequence, biological function and binding characteristics. The amino acid sequence of the latter is highly homologous to the nucleic acid binding domain (i.e. the cold shock domain) of proteins belonging to the Y-box family. Despite their differences in amino acid sequence and function, the folds of the three proteins are remarkably similar, suggesting that this is a preferred folding pattern shared by many single-stranded DNA binding proteins.     

Predicted role for the archease protein family based on structural and sequence analysis of TM1083 and MTH1598, two proteins structurally characterized through structural genomics efforts

Recently, the structures of two proteins belonging to the archease family, TM1083 from Thermotoga maritima and MTH1598 from Methanobacterium thermoautotrophicum, have been solved independently by two Protein Structure Initiative structural genomics pilot centers using X-ray crystallography and NMR, respectively. The archease protein family is a good example of one of the paradoxes of structural genomics: Approximately one third of protein structures produced by structural genomics centers have no known function and are still annotated as "hypothetical proteins" in the Protein Data Bank. In the case of archeases, despite the existence of two protein structures and abundant sequence information, there is still no function assigned to this protein family. Here, our group predicts, based on structural similarity, sequence conservation, and gene context analyses, that members of this protein family might function as chaperones or modulators of proteins involved in DNA/RNA processing. The conservation of genomic context for this protein family is constant from Archaea and Bacteria to humans, and suggests that unannotated open reading frames contiguous to them could be novel RNA/DNA binding proteins.     

Backbone assignments of the 26 kDa neuron-specific ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1)

UCH-L1 is a member of the family of ubiquitin C-terminal hydrolases whose primary role is to hydrolyze small C-terminal adducts of ubiquitin to generate free ubiquitin monomers. Expression of UCH-L1 is highly specific to neurons and point mutations in this enzyme are associated with a hereditary form of Parkinson’s disease. Herein, we present the NMR backbone assignments of human UCH-L1, thus enabling future solution-state NMR spectroscopic studies on the structure and function of this important protein.     

Pactolus I-domain: functional switching of the Rossmann fold

Murine Pactolus is a neutrophil-specific single chain glycoprotein that plays a role as an apoptosis marker for macrophages. The extracellular region of the protein shows strong sequence similarities to integrin beta-subunits. Critical sequence modifications differentiate its function when compared to the integrin family. We show experimentally that Pactolus I-domain does not bind divalent metal ions, indicating that ligand binding is not mediated through a metal ion-dependent adhesion site (MIDAS). NMR data was used to map secondary structure and the strand pairing within the beta-sheet to confirm an overall Rossmann fold topology. Homology modeling enhanced by the NMR data was used to determine the overall structure, with two key loop insertions/deletions (insertion 2 and SDL) that distinguish the Pactolus I-domain from the integrin alpha I-domain and beta I-domains. NMR peak exchange broadening is observed due to dimerization, correlating to the beta I-domain and beta propeller heterodimerization region within the integrin headpiece. Two unique N-linked glycosylation sites (Asn151 and Asn230) within this region disrupt dimerization and may account for why Pactolus is not found to associate with an alpha-subunit. These changes in quaternary structure, ligand binding loops, glycosylation, and metal sites illustrate how evolution has rapidly and effectively altered key aspects of the integrin beta-subunit to derive a protein of novel function on an existing protein scaffold.     

Solution structures of two CCHC zinc fingers from the FOG family protein U-shaped that mediate protein-protein interactions

BACKGROUND: Zinc finger domains have traditionally been regarded as sequence-specific DNA binding motifs. However, recent evidence indicates that many zinc fingers mediate specific protein-protein interactions. For instance, several zinc fingers from FOG family proteins have been shown to interact with the N-terminal zinc finger of GATA-1. RESULTS: We have used NMR spectroscopy to determine the first structures of two FOG family zinc fingers that are involved in protein-protein interactions: fingers 1 and 9 from U-shaped. These fingers resemble classical TFIIIA-like zinc fingers, with the exception of an unusual extended portion of the polypeptide backbone prior to the fourth zinc ligand. [15N,(1)H]-HSQC titrations have been used to define the GATA binding surface of USH-F1, and comparison with other FOG family proteins indicates that the recognition mechanism is conserved across species. The surface of FOG-type fingers that interacts with GATA-1 overlaps substantially with the surface through which classical fingers typically recognize DNA. This suggests that these fingers could not contact both GATA and DNA simultaneously. In addition, results from NMR, gel filtration, and sedimentation equilibrium experiments suggest that the interactions are of moderate affinity. CONCLUSIONS: Our results demonstrate unequivocally that zinc fingers comprising the classical betabetaalpha fold are capable of mediating specific contacts between proteins. The existence of this alternative function has implications for the prediction of protein function from sequence data and for the evolution of protein function.     

Structure of the coat protein-binding domain of the scaffolding protein from a double-stranded DNA virus

Scaffolding proteins are required for high fidelity assembly of most high T number dsDNA viruses such as the large bacteriophages, and the herpesvirus family. They function by transiently binding and positioning the coat protein subunits during capsid assembly. In both bacteriophage P22 and the herpesviruses the extreme scaffold C terminus is highly charged, is predicted to be an amphipathic alpha-helix, and is sufficient to bind the coat protein, suggesting a common mode of action. NMR studies show that the coat protein-binding domain of P22 scaffolding protein exhibits a helix-loop-helix motif stabilized by a hydrophobic core. One face of the motif is characterized by a high density of positive charges that could interact with the coat protein through electrostatic interactions. Results from previous studies with a truncation fragment and the observed salt sensitivity of the assembly process are explained by the NMR structure.     

复苏促进因子研究进展

复苏促进因子是一类广泛存在于结核分枝杆菌等革兰阳性菌中的蛋白质,能促进休眠菌复苏。复苏促进因子的作用机制目前仍然是未知的,然而对此类蛋白的结构域预测及核磁共振分析显示其保守区具有溶菌酶样折叠。我们简要综述了国外对此类蛋白作用机制的研究进展。     

Solution NMR structure of VF0530 from Vibrio fischeri reveals a nucleic acid-binding function

Protein domain family PF09905 (DUF2132) is a family of small domains of unknown function that are conserved in a wide range of bacteria. Here we describe the solution NMR structure of the 80-residue VF0530 protein from Vibrio fischeri, the first structural representative from this protein domain family. We demonstrate that the structure of VF0530 adopts a unique four-helix motif that shows some similarity to the C-terminal double-stranded DNA (dsDNA) binding domain of RecA, as well as other nucleic acid binding domains. Moreover, gel shift binding data indicate a potential dsDNA binding role for VF0530.     

Solution structure of a yeast ubiquitin-like protein Smt3: the role of structurally less defined sequences in protein-protein recognitions

Smt3 belongs to a growing family of ubiquitin-related proteins involved in posttranslational protein modification. Independent studies demonstrate an essential function of Smt3 in the regulation of nucleocytoplasmic transport, and suggest a role in cell-cycle regulation. Here we report the high-resolution NMR structure of yeast Smt3 in the complex free form. Our comparison of the Smt3 NMR structure with the Smt3 crystal structure in complex with the C-Terminal Ulp1 protease domain revealed large structural differences in the binding surface, which is also involved in the Smt3-Ubc-9 interaction detected by NMR. The structural differences in the region indicate the important functions of conserved residues in less structurally defined sequences.     

The cytoplasmic domain of the chloride channel ClC-0: structural and dynamic characterization of flexible regions

Eukaryotic members of the ClC family of chloride channels and transporters are composed of a transmembrane ion transport domain followed by a cytoplasmic domain, which is believed to be involved in the modulation of ClC function. In some family members this putative regulatory domain contains next to a well-folded structured part, long sequence stretches with low sequence complexity. These regions, a 96 residue long linker connecting two structured sub-domains, and 35 residues on the C teminus of the domain were found disordered in a recent crystal structure of this domain in ClC-0. Both regions have a large influence on the modulation of channel function in closely related family members. Here we describe a NMR study to characterize the structural and dynamic properties of these putatively unstructured stretches. Our study reveals that the two regions indeed show large conformational flexibility with dynamics on the nanosecond timescale. However, small islands of secondary structure are found interdispersed between the unfolded regions. This study characterizes for the first time the biophysical properties of these protein segments, which may become important for the understanding of novel regulatory mechanisms within the ClC family.     

Chemical shift assignments of the C-terminal Eps15 homology domain-3 EH domain

The C-terminal Eps15 homology (EH) domain 3 (EHD3) belongs to a eukaryotic family of endocytic regulatory proteins and is involved in the recycling of various receptors from the early endosome to the endocytic recycling compartment or in retrograde transport from the endosomes to the Golgi. EH domains are highly conserved in the EHD family and function as protein-protein interaction units that bind to Asn-Pro-Phe (NPF) motif-containing proteins. The EH domain of EHD1 was the first C-terminal EH domain from the EHD family to be solved by NMR. The differences observed between this domain and proteins with N-terminal EH domains helped describe a mechanism for the differential binding of NPF-containing proteins. Here, structural studies were expanded to include the EHD3 EH domain. While the EHD1 and EHD3 EH domains are highly homologous, they have different protein partners. A comparison of these structures will help determine the selectivity in protein binding between the EHD family members and lead to a better understanding of their unique roles in endocytic regulation.     

The solution structure of antigen MPT64 from Mycobacterium tuberculosis defines a new family of beta-grasp proteins

The MPT64 protein and its homologs form a highly conserved family of secreted proteins with unknown function that are found within the pathogenic Mycobacteria genus. The founding member of this family from Mycobacterium tuberculosis (MPT64 or protein Rv1980c) is expressed only when Mycobacteria cells are actively dividing. By virtue of this relatively unique expression profile, Rv1980c is currently under phase III clinical trials to evaluate its potential to replace tuberculin, or purified protein derivative, as the rapid diagnostic of choice for detection of active tuberculosis infection. We describe here the NMR solution structure of Rv1980c. This structure reveals a previously undescribed fold that is based upon a variation of a beta-grasp motif most commonly found in protein-protein interaction domains. Examination of this structure in conjunction with multiple sequence alignments of MPT64 homologs identifies a candidate ligand-binding site, which may help guide future studies of Rv1980c function. The work presented here also suggests structure-based approaches for increasing the antigenic potency of a Rv1980c-based diagnostic.     

Solution structure of human saposin C: pH-dependent interaction with phospholipid vesicles

Saposin C binds to membranes to activate lipid degradation in lysosomes. To get insights into saposin C's function, we have determined its three-dimensional structure by NMR and investigated its interaction with phospholipid vesicles. Saposin C adopts the saposin-fold common to other members of the family. In contrast, the electrostatic surface revealed by the NMR structure is remarkably different. We suggest that charge distribution in the protein surface can modulate membrane interaction leading to the functional diversity of this family. We find that the binding of saposin C to phospholipid vesicles is a pH-controlled reversible process. The pH dependence of this interaction is sigmoidal, with an apparent pK(a) for binding close to 5.3. The pK(a) values of many solvent-exposed Glu residues are anomalously high and close to the binding pK(a). Our NMR data are consistent with the absence of a conformational change prior to membrane binding. All this information suggests that the negatively charged electrostatic surface of saposin C needs to be partially neutralized to trigger membrane binding. We have studied the membrane-binding behavior of a mutant of saposin C designed to decrease the negative charge of the electrostatic surface. The results support our conclusion on the importance of protein surface neutralization in binding. Since saposin C is a lysosomal protein and pH gradients occur in lysosomes, we propose that lipid degradation in the lysosome could be switched on and off by saposin C's reversible binding to membranes.     

Protocols and pitfalls in obtaining fatty acid-binding proteins for biophysical studies of ligand-protein and protein-protein interactions

Adipocyte fatty acid-binding protein (AFABP: FABP4) is a member of the intracellular lipid-binding protein family that is thought to target long-chain fatty acids to nuclear receptors such as peroxisome proliferator-activated receptor gamma (PPARγ), which in turn plays roles in insulin resistance and obesity. A molecular understanding of AFABP function requires robust isolation of the protein in liganded and free forms as well as characterization of its oligomerization state(s) under physiological conditions. We report development of a protocol to optimize the production of members of this protein family in pure form, including removal of their bound lipids by mixing with hydrophobically functionalized hydroxypropyl dextran beads and validation by two-dimensional NMR spectroscopy. The formation of self-associated or covalently bonded protein dimers was evaluated critically using gel filtration chromatography, revealing conditions that promote or prevent formation of disulfide-linked homodimers. The resulting scheme provides a solid foundation for future investigations of AFABP interactions with key ligand and protein partners involved in lipid metabolism.