Solution-state NMR structure of the putative morphogene protein BolA (PFE0790c) from Plasmodium falciparum.

Protozoa of the genus Plasmodium are responsible for malaria, which is perhaps the most important parasitic disease to infect mankind. The emergence of Plasmodium strains resistant to current therapeutics and prophylactics makes the development of new treatment strategies urgent. Among the potential targets for new antimalarial drugs is the BolA-like protein PFE0790c from Plasmodium falciparum (Pf-BolA).

Structural characterization of a flexible two-domain protein in solution using small angle X-ray scattering and NMR data.

Multidomain proteins in which individual domains are connected by linkers often possess inherent interdomain flexibility that significantly complicates their structural characterization in solution using either nuclear magnetic resonance (NMR) spectroscopy or small-angle X-ray scattering (SAXS) alone. Here, we report a protocol for joint refinement of flexible multidomain protein structures against NMR distance and angular restraints, residual dipolar couplings, and SAXS data. The protocol is based on the ensemble optimization method principle (Bernadó et al.

Structural and functional characterization of DUF1471 domains of Salmonella proteins SrfN, YdgH/SssB, and YahO.

Bacterial species in the Enterobacteriaceae typically contain multiple paralogues of a small domain of unknown function (DUF1471) from a family of conserved proteins also known as YhcN or BhsA/McbA. Proteins containing DUF1471 may have a single or three copies of this domain. Representatives of this family have been demonstrated to play roles in several cellular processes including stress response, biofilm formation, and pathogenesis.

Screening proteins for NMR suitability.

NMR spectroscopy is a valuable tool in structural genomics. Identification of protein samples that are amenable to structure determination by NMR spectroscopy requires efficient screening. The preparation of multiple samples in parallel and screening by NMR is described.

Solution NMR structure and histone binding of the PHD domain of human MLL5.

Mixed Lineage Leukemia 5 (MLL5) is a histone methyltransferase that plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. In addition to its catalytic domain, MLL5 contains a PHD finger domain, a protein module that is often involved in binding to the N-terminus of histone H3. Here we report the NMR solution structure of the MLL5 PHD domain showing a variant of the canonical PHD fold that combines conserved H3 binding features from several classes of other PHD domains (including an aromatic cage) along with a novel C-terminal α-helix, not previously seen.

The ZIP5 ectodomain co-localizes with PrP and may acquire a PrP-like fold that assembles into a dimer.

The cellular prion protein (PrP(C)) was recently observed to co-purify with members of the LIV-1 subfamily of ZIP zinc transporters (LZTs), precipitating the surprising discovery that the prion gene family descended from an ancestral LZT gene. Here, we compared the subcellular distribution and biophysical characteristics of LZTs and their PrP-like ectodomains. When expressed in neuroblastoma cells, the ZIP5 member of the LZT subfamily was observed to be largely directed to the same subcellular locations as PrP(C) and both proteins were seen to be endocytosed through vesicles decorated with the Rab5 marker protein.

Solution NMR structure of hypothetical protein CV_2116 encoded by a viral prophage element in Chromobacterium violaceum.

CV_2116 is a small hypothetical protein of 82 amino acids from the Gram-negative coccobacillus Chromobacterium violaceum. A PSI-BLAST search using the CV_2116 sequence as a query identified only one hit (E = 2e(-07)) corresponding to a hypothetical protein OR16_04617 from Cupriavidus basilensis OR16, which failed to provide insight into the function of CV_2116. The CV_2116 gene was cloned into the p15TvLic expression plasmid, transformed into E.

Structural analysis of HopPmaL reveals the presence of a second adaptor domain common to the HopAB family of Pseudomonas syringae type III effectors.

HopPmaL is a member of the HopAB family of type III effectors present in the phytopathogen Pseudomonas syringae. Using both X-ray crystallography and solution nuclear magnetic resonance, we demonstrate that HopPmaL contains two structurally homologous yet functionally distinct domains. The N-terminal domain corresponds to the previously described Pto-binding domain, while the previously uncharacterised C-terminal domain spans residues 308-385.

Zn-binding AZUL domain of human ubiquitin protein ligase Ube3A.

Ube3A (also referred to as E6AP for E6 Associated Protein) is a E3 ubiquitin-protein ligase implicated in the development of Angelman syndrome by controlling degradation of synaptic protein Arc and oncogenic papilloma virus infection by controlling degradation of p53. This article describe the solution NMR structure of the conserved N-terminal domain of human Ube3A (residues 24-87) that contains two residues (Cys44 and Arg62) found to be mutated in patients with Angelman syndrome. The structure of this domain adopts a novel Zn-binding fold we called AZUL (Amino-terminal Zn-finger of Ube3a Ligase).

NleG Type 3 effectors from enterohaemorrhagic Escherichia coli are U-Box E3 ubiquitin ligases.

NleG homologues constitute the largest family of Type 3 effectors delivered by pathogenic E. coli, with fourteen members in the enterohaemorrhagic (EHEC) O157:H7 strain alone. Identified recently as part of the non-LEE-encoded (Nle) effector set, this family remained uncharacterised and shared no sequence homology to other proteins including those of known function.

The solution structure of ribosomal protein S17E from Methanobacterium thermoautotrophicum: a structural homolog of the FF domain.

The ribosomal protein S17E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. S17E is a 62-residue protein conserved in archaea and eukaryotes and has no counterparts in bacteria. Mammalian S17E is a phosphoprotein component of eukaryotic ribosomes.

Solution structure of ribosomal protein L40E, a unique C4 zinc finger protein encoded by archaeon Sulfolobus solfataricus.

The ribosomal protein L40E from archaeon Sulfolobus solfataricus is a component of the 50S ribosomal subunit. L40E is a 56-residue, highly basic protein that contains a C4 zinc finger motif, CRKC_X(10)_CRRC. Homologs are found in both archaea and eukaryotes but are not present in bacteria.

The hypothetical protein Atu4866 from Agrobacterium tumefaciens adopts a streptavidin-like fold.

Atu4866 is a 79-residue conserved hypothetical protein of unknown function from Agrobacterium tumefaciens. Protein sequence alignments show that it shares > or =60% sequence identity with 20 other hypothetical proteins of bacterial origin. However, the structures and functions of these proteins remain unknown so far.

In situ proteolysis for protein crystallization and structure determination.

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

Solution structure of the hypothetical protein TA0095 from Thermoplasma acidophilum: a novel superfamily with a two-layer sandwich architecture.

TA0095 is a 96-residue hypothetical protein from Thermoplasma acidophilum that exhibits no sequence similarity to any protein of known structure. Also, TA0095 is a member of the COG4004 orthologous group of unknown function found in Archaea bacteria. We determined its three-dimensional structure by NMR methods.

Hypothetical protein AF2241 from Archaeoglobus fulgidus adopts a cyclophilin-like fold.

AF2241 is a hypothetical protein from Archaeoglobus fulgidus and it belongs to the PFam domain of unknown function 369 (DUF369). NMR structural determination reveals that AF2241 adopts a cyclophilin-like fold, with a beta-barrel core composed of eight beta-strands, one alpha-helix, and one 3(10) helix located at each end of the barrel. The protein displays a high structural similarity to TM1367, another member of DUF369 whose structure has been determined recently by X-ray crystallography.

NMR structure and binding studies confirm that PA4608 from Pseudomonas aeruginosa is a PilZ domain and a c-di-GMP binding protein.

PA4608 is a 125 residue protein from Pseudomonas aeruginosa with a recent identification as a PilZ domain and putative bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) adaptor protein that plays a role in bacterial second-messenger regulated processes. The nuclear magnetic resonance (NMR) structure of PA4608 has been determined and c-di-GMP binding has been confirmed by NMR titration studies. The monomeric core structure of PA4608 contains a six-stranded anti-parallel beta barrel flanked by three helices.