Solution NMR structures reveal a distinct architecture and provide first structures for protein domain family PF04536

The protein family (Pfam) PF04536 is a broadly conserved domain family of unknown function (DUF477), with more than 1,350 members in prokaryotic and eukaryotic proteins. High-quality NMR structures of the N-terminal domain comprising residues 41–180 of the 684-residue protein CG2496 from Corynebacterium glutamicum and the N-terminal domain comprising residues 35–182 of the 435-residue protein PG0361 from Porphyromonas gingivalis both exhibit an α/β fold comprised of a four-stranded β-sheet, three α-helices packed against one side of the sheet, and a fourth α-helix attached to the other side. In spite of low sequence similarity (18%) assessed by structure-based sequence alignment, the two structures are globally quite similar. However, moderate structural differences are observed for the relative orientation of two of the four helices. Comparison with known protein structures reveals that the α/β architecture of CG2496(41–180) and PG0361(35–182) has previously not been characterized. Moreover, calculation of surface charge potential and identification of surface clefts indicate that the two domains very likely have different functions.     

Solution NMR structures provide first structural coverage of the large protein domain family PF08369 and complementary structural coverage of dark operative protochlorophyllide oxidoreductase complexes

High-quality NMR structures of the C-terminal domain comprising residues 484–537 of the 537-residue protein Bacterial chlorophyll subunit B (BchB) from Chlorobium tepidum and residues 9–61 of 61-residue Asr4154 from Nostoc sp. (strain PCC 7120) exhibit a mixed α/β fold comprised of three α-helices and a small β-sheet packed against second α-helix. These two proteins share 29 % sequence similarity and their structures are globally quite similar. The structures of BchB(484–537) and Asr4154(9–61) are the first representative structures for the large protein family (Pfam) PF08369, a family of unknown function currently containing 610 members in bacteria and eukaryotes. Furthermore, BchB(484–537) complements the structural coverage of the dark-operating protochlorophyllide oxidoreductase.     

Solution NMR and X-ray crystal structures of membrane-associated Lipoprotein-17 domain reveal a novel fold

The conserved Lipoprotein-17 domain of membrane-associated protein Q9PRA0_UREPA from Ureaplasma parvum was selected for structure determination by the Northeast Structural Genomics Consortium, as part of the Protein Structure Initiative's program on structure-function analysis of protein domains from large domain sequence families lacking structural representatives. The 100-residue Lipoprotein-17 domain is a "domain of unknown function" (DUF) that is a member of Pfam protein family PF04200, a large domain family for which no members have characterized biochemical functions. The three-dimensional structure of the Lipoprotein-17 domain of protein Q9PRA0_UREPA was determined by both solution NMR and by X-ray crystallography at 2.5 ?. The two structures are in good agreement with each other. The domain structure features three α-helices, α1 through α3, and five β-strands. Strands β1/β2, β3/β4, β4/β5 are anti-parallel to each other. Strands β1and β2 are orthogonal to strands β3, β4, β5, while helix α3 is formed between the strands β3 and β4. One-turn helix α2 is formed between the strands β1 and β2, while helix α1 occurs in the N-terminal polypeptide segment. Searches of the Protein Data Bank do not identify any other protein with significant structural similarity to Lipoprotein-17 domain of Q9PRA0_UREPA, indicating that it is a novel protein fold.     

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 NMR structure of Dsy0195 homodimer from Desulfitobacterium hafniense: first structure representative of the YabP domain family of proteins involved in spore coat assembly

Protein domain family YabP (PF07873) is a family of small protein domains that are conserved in a wide range of bacteria and involved in spore coat assembly during the process of sporulation. The 62-residue fragment of Dsy0195 from Desulfitobacterium hafniense, which belongs to the YabP family, exists as a homodimer in solution under the conditions used for structure determination using NMR spectroscopy. The structure of the Dsy0195 homodimer contains two identical 62-residue monomeric subunits, each consisting of five anti-parallel beta strands (β1, 23-29; β2, 31-38; β3, 41-46; β4, 49-59; β5, 69-80). The tertiary structure of the Dsy0195 monomer adopts a cylindrical fold composed of two beta sheets. The two monomer subunits fold into a homodimer about a single C2 symmetry axis, with the interface composed of two anti-parallel beta strands, β1-β1' and β5b-β5b', where β5b refers to the C-terminal half of the bent β5 strand, without any domain swapping. Potential functional regions of the Dsy0195 structure were predicted based on conserved sequence analysis. The Dsy0195 structure reported here is the first representative structure from the YabP family.     

Solution NMR structures of the C‐domain of Tetrahymena cytoskeletal protein Tcb2 reveal distinct calcium‐induced structural rearrangements

Tcb2 is a calcium‐binding protein that localizes to the membrane‐associated skeleton of the ciliated protozoan Tetrahymena thermophila with hypothesized roles in ciliary movement, cell cortex signaling, and pronuclear exchange. Tcb2 has also been implicated in a unique calcium‐triggered, ATP‐independent type of contractility exhibited by filamentous networks isolated from the Tetrahymena cytoskeleton. To gain insight into Tcb2's structure‐function relationship and contractile properties, we determined solution NMR structures of its C‐terminal domain in the calcium‐free and calcium‐bound states. The overall architecture is similar to other calcium‐binding proteins, with paired EF‐hand calcium‐binding motifs. Comparison of the two structures reveals that Tcb2‐C's calcium‐induced conformational transition differs from the prototypical calcium sensor calmodulin, suggesting that the two proteins play distinct functional roles in Tetrahymena and likely have different mechanisms of target recognition. Future studies of the full‐length protein and the identification of Tcb2 cellular targets will help establish the molecular basis of Tcb2 function and its unique contractile properties. Proteins 2016; 84:1748–1756. © 2016 Wiley Periodicals, Inc.     

NMR structures of the selenoproteins Sep15 and SelM reveal redox activity of a new thioredoxin-like family

Selenium has significant health benefits, including potent cancer prevention activity and roles in immune function and the male reproductive system. Selenium-containing proteins, which incorporate this essential micronutrient as selenocysteine, are proposed to mediate the positive effects of dietary selenium. Presented here are the solution NMR structures of the selenoprotein SelM and an ortholog of the selenoprotein Sep15. These data reveal that Sep15 and SelM are structural homologs that establish a new thioredoxin-like protein family. The location of the active-site redox motifs within the fold together with the observed localized conformational changes after thiol-disulfide exchange and measured redox potential indicate that they have redox activity. In mammals, Sep15 expression is regulated by dietary selenium, and either decreased or increased expression of this selenoprotein alters redox homeostasis. A physiological role for Sep15 and SelM as thiol-disulfide oxidoreductases and their contribution to the quality control pathways of the endoplasmic reticulum are discussed.     

Structure of the N-terminal oligomerization domain of DnaD reveals a unique tetramerization motif and provides insights into scaffold formation

DnaD is a primosomal protein that remodels supercoiled plasmids. It binds to supercoiled forms and converts them to open forms without nicking. During this remodeling process, all the writhe is converted to twist and the plasmids are held around the periphery of large scaffolds made up of DnaD molecules. This DNA-remodeling function is the sum of a scaffold-forming activity on the N-terminal domain and a DNA-dependent oligomerization activity on the C-terminal domain. We have determined the crystal structure of the scaffold-forming N-terminal domain, which reveals a winged-helix architecture, with additional structural elements extending from both N- and C-termini. Four monomers form dimers that join into a tetramer. The N-terminal extension mediates dimerization and tetramerization, with extensive interactions and distinct interfaces. The wings and helices of the winged-helix domains remain exposed on the surface of the tetramer. Structure-guided mutagenesis and atomic force microscopy imaging indicate that these elements, together with the C-terminal extension, are involved in scaffold formation. Based upon our data, we propose a model for the DnaD-mediated scaffold formation.     

Secondary structure determination for the Antennapedia homeodomain by nuclear magnetic resonance and evidence for a helix-turn-helix motif.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was studied in aqueous solution by nuclear magnetic resonance (NMR). Sequence-specific resonance assignments have been obtained for the complete polypeptide chain of 68 amino acid residues. The secondary structure determined from nuclear Overhauser effects (NOE) and information about slowly exchanging amide protons includes three helical segments consisting of the residues 10-21, 28-38 and 42-52, respectively. Combination of the presently available NMR data with computer modeling provided preliminary evidence for the presence of a helix-turn-helix motif in the homeodomain. Near the turn, this supersecondary structure appears to be very similar to the DNA binding site in the 434 and P22 c2 repressors, but both helices in the homeodomain include 2-3 additional residues when compared with these prokaryotic DNA-binding proteins.     

The formation of viroplasm-like structures by the rotavirus NSP5 protein is calcium regulated and directed by a C-terminal helical domain

The rotavirus NSP5 protein directs the formation of viroplasm-like structures (VLS) and is required for viroplasm formation within infected cells. In this report, we have defined signals within the C-terminal 21 amino acids of NSP5 that are required for VLS formation and that direct the insolubility and hyperphosphorylation of NSP5. Deleting C-terminal residues of NSP5 dramatically increased the solubility of N-terminally tagged NSP5 and prevented NSP5 hyperphosphorylation. Computer modeling and analysis of the NSP5 C terminus revealed the presence of an amphipathic alpha-helix spanning 21 C-terminal residues that is conserved among rotaviruses. Proline-scanning mutagenesis of the predicted helix revealed that single-amino-acid substitutions abolish NSP5 insolubility and hyperphosphorylation. Helix-disrupting NSP5 mutations also abolished localization of green fluorescent protein (GFP)-NSP5 fusions into VLS and directly correlate VLS formation with NSP5 insolubility. All mutations introduced into the hydrophobic face of the predicted NSP5 alpha-helix disrupted VLS formation, NSP5 insolubility, and the accumulation of hyperphosphorylated NSP5 isoforms. Some NSP5 mutants were highly soluble but still were hyperphosphorylated, indicating that NSP5 insolubility was not required for hyperphosphorylation. Expression of GFP containing the last 68 residues of NSP5 at its C terminus resulted in the formation of punctate VLS within cells. Interestingly, GFP-NSP5-C68 was diffusely dispersed in the cytoplasm when calcium was depleted from the medium, and after calcium resupplementation GFP-NSP5-C68 rapidly accumulated into punctate VLS. A potential calcium switch, formed by two tandem pseudo-EF-hand motifs (DxDxD), is present just upstream of the predicted alpha-helix. Mutagenesis of either DxDxD motif abolished the regulatory effect of calcium on VLS formation and resulted in the constitutive assembly of GFP-NSP5-C68 into punctate VLS. These results reveal specific residues within the NSP5 C-terminal domain that direct NSP5 hyperphosphorylation, insolubility, and VLS formation in addition to defining residues that constitute a calcium-dependent trigger of VLS formation. These studies identify functional determinants within the C terminus of NSP5 that regulate VLS formation and provide a target for inhibiting NSP5-directed VLS functions during rotavirus replication.     

Chemically accurate protein structures: Validation of protein NMR structures by comparison of measured and predicted pK a values

A new method is presented for evaluating the quality of protein structures obtained by NMR. This method exploits the dependence between measurable chemical properties of a protein, namely pK _a values of acidic residues, and protein structure. The accurate and fast empirical computational method employed by the PROPKA program () allows the user to test the ability of a given structure to reproduce known pK _a values, which in turn can be used as a criterion for the selection of more accurate structures. We demonstrate the feasibility of this novel idea for a series of proteins for which both␣NMR and X-ray structures, as well as pK _a values of all ionizable residues, have been determined. For the 17 NMR ensembles used in this study, this criterion is shown effective in the elimination of a large number of NMR structure ensemble members.     

Solution structure of the first RNA recognition motif domain of human spliceosomal protein SF3b49 and its mode of interaction with a SF3b145 fragment

The spliceosomal protein SF3b49, a component of the splicing factor 3b (SF3b) protein complex in the U2 small nuclear ribonucleoprotein, contains two RNA recognition motif (RRM) domains. In yeast, the first RRM domain (RRM1) of Hsh49 protein (yeast orthologue of human SF3b49) reportedly interacts with another component, Cus1 protein (orthologue of human SF3b145). Here, we solved the solution structure of the RRM1 of human SF3b49 and examined its mode of interaction with a fragment of human SF3b145 using NMR methods. Chemical shift mapping showed that the SF3b145 fragment spanning residues 598–631 interacts with SF3b49 RRM1, which adopts a canonical RRM fold with a topology of β1‐α1‐β2‐β3‐α2‐β4. Furthermore, a docking model based on NOESY measurements suggests that residues 607–616 of the SF3b145 fragment adopt a helical structure that binds to RRM1 predominantly via α1, consequently exhibiting a helix–helix interaction in almost antiparallel. This mode of interaction was confirmed by a mutational analysis using GST pull‐down assays. Comparison with structures of all RRM domains when complexed with a peptide found that this helix–helix interaction is unique to SF3b49 RRM1. Additionally, all amino acid residues involved in the interaction are well conserved among eukaryotes, suggesting evolutionary conservation of this interaction mode between SF3b49 RRM1 and SF3b145.     

NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+ -binding motif

Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of glial precursors from the neural default to glia. The GCM protein contains the functional domain that is essential for recognition of the upstream sequence of the repo gene. In the DNA-binding region of this GCM protein, there is a cysteine-rich region with which divalent metal ions such as Zn(2+) must bind and other proteins belonging to the GCM family have a corresponding region. To obtain a more detailed insight into the structural and functional features of this DNA-binding region, we have determined the minimal DNA-binding domain and obtained inductively coupled plasma atomic emission spectra and (1)H-(15)N, (1)H-(15)N-(13)C and (113)Cd(2+) NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn(2+)-binding sites, one of which is adjacent to the interface for DNA binding. Systematic mutational analyses of the conserved cysteine residues in the minimal DNA-binding domain revealed that one Zn(2+)-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.