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1.  Hydrodynamic characterization of recombinant human fibrinogen species 
Thrombosis research  2013;132(1):e48-e53.
Introduction
Fibrinogen is a key component of the blood coagulation system and plays important, diverse roles in several relevant pathologies such as thrombosis, hemorrhage, and cancer. It is a large glycoprotein whose three-dimensional molecular structure is not fully known. Furthermore, circulating fibrinogen is highly heterogeneous, mainly due to proteolytic degradation and alternative mRNA processing. Recombinant production of human fibrinogen allows investigating the impact on the three-dimensional structure of specific changes in the primary structure.
Methods
We performed analytical ultracentrifugation analyses of a full-length recombinant human fibrinogen, its counterpart purified from human plasma, and a recombinant human fibrinogen with both Aα chains truncated at amino acid 251, thus missing their last 359 amino acid residues.
Results
We have accurately determined the translational diffusion and sedimentation coefficients (Dt(20,w)0, s(20,w)0) of all three species. This was confirmed by derived molecular weights within 1% for the full length species, and 5% for the truncated species, as assessed by comparison with SDS-PAGE/Western blot analyses and primary structure data. No significant differences in the values of Dt(20,w)0 and s(20,w)0 were found between the recombinant and purified full length human fibrinogens, while slightly lower and higher values, respectively, resulted for the recombinant truncated human fibrinogen compared to a previously characterized purified human fibrinogen fragment X obtained by plasmin digestion.
Conclusions
Full-length recombinant fibrinogen is less polydisperse but hydrodynamically indistinguishable from its counterpart purified from human plasma. Recombinant Aα251-truncated human fibrinogen instead behaves differently from fragment X, suggesting a role for the Bβ residues 1–52 in inter-molecular interactions. Overall, these new hydrodynamic data will constitute a reliable benchmark against which models of fibrinogen species could be compared.
doi:10.1016/j.thromres.2013.04.005
PMCID: PMC3742573  PMID: 23642654
Recombinant Fibrinogen; Blood Coagulation; Analytical Ultracentrifugation; Hydrodynamics
2.  Assembly of the Respiratory Mucin MUC5B 
The Journal of Biological Chemistry  2014;289(23):16409-16420.
Background: Mucin polymer formation is a complex intracellular process.
Results: MUC5B N-terminal D3-domains form reversible pH-sensitive calcium mediated cross-links between linear MUC5B polymer chains.
Conclusion: Intracellular assembly of MUC5B generates disulfide-bonded polymers which form calcium mediated condensed networks in secretory granules.
Significance: This identifies a new model for mucin assembly that may be common to other polymeric mucins.
Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer. Self-diffusion studies with intact MUC5B showed that calcium binding at the protein site catalyzed reversible cross-links between MUC5B chains to form networks. The site of cross-linking was identified in the MUC5B D3-domain as it was specifically blocked by D3 peptide antibodies. Biophysical analysis and single particle EM of recombinant MUC5B N terminus (D1D2D′D3; NT5B) and subdomains (D1, D1-D2, D2-D′-D3, and D3) generated structural models of monomers and disulfide-linked dimers and suggested that MUC5B multimerizes by disulfide linkage between D3-domains to form linear polymer chains. Moreover, these analyses revealed reversible homotypic interactions of NT5B at low pH and in high calcium, between disulfide-linked NT5B dimers, but not monomers. These results enable a model of MUC5B to be derived, which predicts mechanisms of mucin intracellular assembly and storage, which may be common to the other major gel-forming polymeric mucins.
doi:10.1074/jbc.M114.566679
PMCID: PMC4047408  PMID: 24778189
Analytical Ultracentrifugation; Cystic Fibrosis; Mucin; Mucus; Recombinant Protein Expression; Single Particle Analysis; Goblet Cell
3.  Interaction of a Partially Disordered Antisigma Factor with Its Partner, the Signaling Domain of the TonB-Dependent Transporter HasR 
PLoS ONE  2014;9(4):e89502.
Bacteria use diverse signaling pathways to control gene expression in response to external stimuli. In Gram-negative bacteria, the binding of a nutrient is sensed by an outer membrane transporter. This signal is then transmitted to an antisigma factor and subsequently to the cytoplasm where an ECF sigma factor induces expression of genes related to the acquisition of this nutrient. The molecular interactions involved in this transmembrane signaling are poorly understood and structural data on this family of antisigma factor are rare. Here, we present the first structural study of the periplasmic domain of an antisigma factor and its interaction with the transporter. The study concerns the signaling in the heme acquisition system (Has) of Serratia marcescens. Our data support unprecedented partially disordered periplasmic domain of an anti-sigma factor HasS in contact with a membrane-mimicking environment. We solved the 3D structure of the signaling domain of HasR transporter and identified the residues at the HasS−HasR interface. Their conservation in several bacteria suggests wider significance of the proposed model for the understanding of bacterial transmembrane signaling.
doi:10.1371/journal.pone.0089502
PMCID: PMC3984077  PMID: 24727671
5.  The Carboxy-Terminal αN Helix of the Archaeal XerA Tyrosine Recombinase Is a Molecular Switch to Control Site-Specific Recombination 
PLoS ONE  2013;8(5):e63010.
Tyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding.
Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.
doi:10.1371/journal.pone.0063010
PMCID: PMC3646895  PMID: 23667562
6.  The α-helical regions of KERP1 are important in Entamoeba histolytica adherence to human cells 
Scientific Reports  2013;3:1171.
The lysine and glutamic acid rich protein KERP1 is a unique surface adhesion factor associated with virulence in the human pathogen Entamoeba histolytica. Both the function and structure of this protein remain unknown to this date. Here, we used circular dichroism, analytical ultracentrifugation and bioinformatics modeling to characterize the structure of KERP1. Our findings revealed that it is an α-helical rich protein organized as a trimer, endowed with a very high thermal stability (Tm = 89.6°C). Bioinformatics sequence analyses and 3D-structural modeling indicates that KERP1 central segments could account for protein trimerization. Relevantly, expressing the central region of KERP1 in living parasites, impair their capacity to adhere to human cells. Our observations suggest a link between the inhibitory effect of the isolated central region and the structural features of KERP1.
doi:10.1038/srep01171
PMCID: PMC3558696  PMID: 23378906
7.  Structural Basis for the ABO Blood-Group Dependence of Plasmodium falciparum Rosetting 
PLoS Pathogens  2012;8(7):e1002781.
The ABO blood group influences susceptibility to severe Plasmodium falciparum malaria. Recent evidence indicates that the protective effect of group O operates by virtue of reduced rosetting of infected red blood cells (iRBCs) with uninfected RBCs. Rosetting is mediated by a subgroup of PfEMP1 adhesins, with RBC binding being assigned to the N-terminal DBL1α1 domain. Here, we identify the ABO blood group as the main receptor for VarO rosetting, with a marked preference for group A over group B, which in turn is preferred to group O RBCs. We show that recombinant NTS-DBL1α1 and NTS-DBL1α1-CIDR1γ reproduce the VarO-iRBC blood group preference and document direct binding to blood group trisaccharides by surface plasmon resonance. More detailed RBC subgroup analysis showed preferred binding to group A1, weaker binding to groups A2 and B, and least binding to groups Ax and O. The 2.8 Å resolution crystal structure of the PfEMP1-VarO Head region, NTS-DBL1α1-CIDR1γ, reveals extensive contacts between the DBL1α1 and CIDR1γ and shows that the NTS-DBL1α1 hinge region is essential for RBC binding. Computer docking of the blood group trisaccharides and subsequent site-directed mutagenesis localized the RBC-binding site to the face opposite to the heparin-binding site of NTS-DBLα1. RBC binding involves residues that are conserved between rosette-forming PfEMP1 adhesins, opening novel opportunities for intervention against severe malaria. By deciphering the structural basis of blood group preferences in rosetting, we provide a link between ABO blood grouppolymorphisms and rosette-forming adhesins, consistent with the selective role of falciparum malaria on human genetic makeup.
Author Summary
Rosetting, the capacity of infected red blood cells (RBCs) to bind uninfected RBCs, is a Plasmodium falciparum virulence factor. Rosetting is influenced by the ABO blood group, being less efficient with O RBCs. Although this preference may account for protection against severe malaria afforded by the O blood group, its understanding is fragmentary. We identify the ABO blood group as the main receptor for the rosetting Palo Alto VarO parasites, which display a marked preference for blood group A. Rosetting is caused by a sub-group of PfEMP1 adhesins. PfEMP1-VarO shares with other rosetting lines a specific NTS-DBL1α1-CIDR1γ Head region. We show that the Head region binds RBCs more efficiently than NTS-DBL1α1 and that ABO blood group polymorphisms influence binding of both domains. The 2.8 Å resolution crystal structure of the Head region reveals extensive contacts between the DBL1α1 and CIDR1γ domains, and shows structural features of the NTS-DBL1α1 hinge region essential for RBC binding. We localize the RBC-binding site to the face opposite to the heparin-binding site of NTS-DBL1α1 and document direct binding of the Head region to A and B trisaccharides These findings provide novel insights into the interactions established by malaria parasites with a prominent human blood group.
doi:10.1371/journal.ppat.1002781
PMCID: PMC3395597  PMID: 22807674
8.  Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2 
BMC Biochemistry  2011;12:61.
Background
Caseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. The Mycobacterium tuberculosis (MTB) genome contains two genes coding for putative ClpPs, ClpP1 and ClpP2 respectively, that are likely to play a role in the virulence of the bacterium.
Results
We report the first biochemical characterization of ClpP1 and ClpP2 peptidases from MTB. Both proteins were produced and purified in Escherichia coli. Use of fluorogenic model peptides of diverse specificities failed to show peptidase activity with recombinant mycobacterial ClpP1 or ClpP2. However, we found that ClpP1 had a proteolytic activity responsible for its own cleavage after the Arg8 residue and cleavage of ClpP2 after the Ala12 residue. In addition, we showed that the absence of any peptidase activity toward model peptides was not due to an obstruction of the entry pore by the N-terminal flexible extremity of the proteins, nor to an absolute requirement for the ClpX or ClpC ATPase complex. Finally, we also found that removing the putative propeptides of ClpP1 and ClpP2 did not result in cleavage of model peptides.
We have also shown that recombinant ClpP1 and ClpP2 do not assemble in the conventional functional tetradecameric form but in lower order oligomeric species ranging from monomers to heptamers. The concomitant presence of both ClpP1 and ClpP2 did not result in tetradecameric assembly. Deleting the amino-terminal extremity of ClpP1 and ClpP2 (the putative propeptide or entry gate) promoted the assembly in higher order oligomeric species, suggesting that the flexible N-terminal extremity of mycobacterial ClpPs participated in the destabilization of interaction between heptamers.
Conclusion
Despite the conservation of a Ser protease catalytic triad in their primary sequences, mycobacterial ClpP1 and ClpP2 do not have conventional peptidase activity toward peptide models and display an unusual mechanism of self-assembly. Therefore, the mechanism underlying their peptidase and proteolytic activities might differ from that of other ClpP proteolytic complexes.
doi:10.1186/1471-2091-12-61
PMCID: PMC3258218  PMID: 22132756
9.  The orientation of the C-terminal domain of the Saccharomyces cerevisiae Rap1 protein is determined by its binding to DNA 
Nucleic Acids Research  2011;40(7):3197-3207.
Rap1 is an essential DNA-binding factor from the yeast Saccharomyces cerevisiae involved in transcription and telomere maintenance. Its binding to DNA targets Rap1 at particular loci, and may optimize its ability to form functional macromolecular assemblies. It is a modular protein, rich in large potentially unfolded regions, and comprising BRCT, Myb and RCT well-structured domains. Here, we present the architectures of Rap1 and a Rap1/DNA complex, built through a step-by-step integration of small angle X-ray scattering, X-ray crystallography and nuclear magnetic resonance data. Our results reveal Rap1 structural adjustment upon DNA binding that involves a specific orientation of the C-terminal (RCT) domain with regard to the DNA binding domain (DBD). Crystal structure of DBD in complex with a long DNA identifies an essential wrapping loop, which constrains the orientation of the RCT and affects Rap1 affinity to DNA. Based on our structural information, we propose a model for Rap1 assembly at telomere.
doi:10.1093/nar/gkr1166
PMCID: PMC3326314  PMID: 22139930
10.  A S-adenosylmethionine methyltransferase-like domain within the essential, Fe-S-containing yeast protein Dre2 
The Febs Journal  2012;279(12):2108-2119.
Yeast Dre2 is an essential Fe-S cluster-containing protein that has been implicated in cytosolic Fe-S protein biogenesis and in cell death regulation in response to oxidative stress. Its absence in yeast can be complemented by the human homologous antiapoptotic protein cytokine-induced apoptosis inhibitor 1 (also known as anamorsin), suggesting at least one common function. Using complementary techniques, we have investigated the biochemical and biophysical properties of Dre2. We show that it contains an N-terminal domain whose structure in solution consists of a stable well-structured monomer with an overall typical S-adenosylmethionine methyltransferase fold lacking two α-helices and a β-strand. The highly conserved C-terminus of Dre2, containing two Fe-S clusters, influences the flexibility of the N-terminal domain. We discuss the hypotheses that the activity of the N-terminal domain could be modulated by the redox activity of Fe-S clusters containing the C-terminus domain in vivo.
doi:10.1111/j.1742-4658.2012.08597.x
PMCID: PMC3440578  PMID: 22487307
Dre2; iron-sulfur cluster; NMR; SAM methyltransferase fold; yeast

Results 1-10 (10)