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1.  New Insights into Histidine Triad Proteins: Solution Structure of a Streptococcus pneumoniae PhtD Domain and Zinc Transfer to AdcAII 
PLoS ONE  2013;8(11):e81168.
Zinc (Zn2+) homeostasis is critical for pathogen host colonization and invasion. Polyhistidine triad (Pht) proteins, located at the surface of various streptococci, have been proposed to be involved in Zn2+ homeostasis. The phtD gene, coding for a Zn2+-binding protein, is organized in an operon with adcAII coding for the extracellular part of a Zn2+ transporter. In the present work, we investigate the relationship between PhtD and AdcAII using biochemical and structural biology approaches. Immuno-precipitation experiments on purified membranes of Streptococcus pneumoniae (S. pneumoniae) demonstrate that native PhtD and AdcAII interact in vivo confirming our previous in vitro observations. NMR was used to demonstrate Zn2+ transfer from the Zn2+-bound form of a 137 amino acid N-terminal domain of PhtD (t-PhtD) to AdcAII. The high resolution NMR structure of t-PhtD shows that Zn2+ is bound in a tetrahedral site by histidines 83, 86, and 88 as well as by glutamate 63. Comparison of the NMR parameters measured for apo- and Zn2+-t-PhtD shows that the loss of Zn2+ leads to a diminished helical propensity at the C-terminus and increases the local dynamics and overall molecular volume. Structural comparison with the crystal structure of a 55-long fragment of PhtA suggests that Pht proteins are built from short repetitive units formed by three β-strands containing the conserved HxxHxH motif. Taken together, these results support a role for S. pneumoniae PhtD as a Zn2+ scavenger for later release to the surface transporter AdcAII, leading to Zn2+ uptake.
doi:10.1371/journal.pone.0081168
PMCID: PMC3842936  PMID: 24312273
2.  A gp41 MPER-specific Llama VHH Requires a Hydrophobic CDR3 for Neutralization but not for Antigen Recognition 
PLoS Pathogens  2013;9(3):e1003202.
The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.
Author Summary
Due to the absence of an effective vaccine or cure for acquired immunodeficiency syndrome (AIDS), HIV-1 infections still result in high mortality. Two antibodies, 2F5 and 4E10, previously isolated from HIV-1 infected patients, prevent infections by binding to the MPER of gp41, a part of the virus that is difficult to access and only transiently exposed. Here, we immunized llamas with a gp41-based immunogen and subsequently isolated a small antibody fragment (VHH) that can easily access and recognize the MPER. We showed that a unit of two VHH, named bi-2H10, was indeed capable of preventing HIV-1 from infecting cells. We determined the three dimensional structure of the VHH and mapped its interaction site to an MPER region that overlaps with the 2F5 epitope. The 2H10 VHH displays a membrane binding component important for neutralization that resembles that of 2F5. In conclusion, we have developed an immunogen and a small antibody that may have great potential for development of novel anti-HIV/AIDS vaccines and treatments.
doi:10.1371/journal.ppat.1003202
PMCID: PMC3591319  PMID: 23505368
3.  Biochemical and Structural Characterization of the Subclass B1 Metallo-β-Lactamase VIM-4 ▿  
The metallo-β-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii, was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn2+ at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn2+ ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure.
doi:10.1128/AAC.01486-09
PMCID: PMC3067066  PMID: 21149620
4.  Simultaneous use of solution, solid-state NMR and X-ray crystallography to study the conformational landscape of the Crh protein during oligomerization and crystallization 
We explore, using the Crh protein dimer as a model, how information from solution NMR, solid-state NMR and X-ray crystallography can be combined using structural bioinformatics methods, in order to get insights into the transition from solution to crystal. Using solid-state NMR chemical shifts, we filtered intra-monomer NMR distance restraints in order to keep only the restraints valid in the solid state. These filtered restraints were added to solid-state NMR restraints recorded on the dimer state to sample the conformational landscape explored during the oligomerization process. The use of non-crystallographic symmetries then permitted the extraction of converged conformers subsets. Ensembles of NMR and crystallographic conformers calculated independently display similar variability in monomer orientation, which supports a funnel shape for the conformational space explored during the solution-crystal transition. Insights into alternative conformations possibly sampled during oligomerization were obtained by analyzing the relative orientation of the two monomers, according to the restraint precision. Molecular dynamics simulations of Crh confirmed the tendencies observed in NMR conformers, as a paradoxical increase of the distance between the two β1a strands, when the structure gets closer to the crystallographic structure, and the role of water bridges in this context.
PMCID: PMC3170007  PMID: 21918624
structural bioinformatics; NMR structure calculation; ARIA; non-crystallographic symmetry; crystallographic ensemble refinement; molecular dynamics simulation

Results 1-4 (4)