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New insights into histidine triad proteins: solution structure of a Streptococcus pneumoniae PhtD domain and zinc transfer to AdcAII.

ABSTRACT: Zinc (Zn(2+)) 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 Zn(2+) homeostasis. The phtD gene, coding for a Zn(2+)-binding protein, is organized in an operon with adcAII coding for the extracellular part of a Zn(2+) 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 Zn(2+) transfer from the Zn(2+)-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 Zn(2+) 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 Zn(2+)-t-PhtD shows that the loss of Zn(2+) 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 ?-strands containing the conserved HxxHxH motif. Taken together, these results support a role for S. pneumoniae PhtD as a Zn(2+) scavenger for later release to the surface transporter AdcAII, leading to Zn(2+) uptake.

SUBMITTER: Bersch B

PROVIDER: S-EPMC3842936 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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New insights into histidine triad proteins: solution structure of a Streptococcus pneumoniae PhtD domain and zinc transfer to AdcAII.

Bersch Beate B Bougault Catherine C Roux Laure L Favier Adrien A Vernet Thierry T Durmort Claire C

PloS one 20131128 11

Zinc (Zn(2+)) 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 Zn(2+) homeostasis. The phtD gene, coding for a Zn(2+)-binding protein, is organized in an operon with adcAII coding for the extracellular part of a Zn(2+) transporter. In the present work, we investigate the relationship between PhtD and AdcAII using biochemical and structural biology approache ...[more]

PMID: 24312273

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Project description:IntroductionPneumococcus is an important respiratory pathogen that is associated with high rates of death in newborn children and the elderly. Given the disadvantages of current polysaccharide-based vaccines, the most promising alternative for developing improved vaccines may be to use protein antigens with different roles in pneumococcus virulence. PspA and PhtD, highly immunogenic surface proteins expressed by almost all pneumococcal strains, are capable of eliciting protective immunity against lethal infections.MethodsIn this study using immunoinformatics approaches, we constructed one fusion construct (called PAD) by fusing the immunodominant regions of PspA from families 1 & 2 (PA) to the immunodominant regions of PhtD (PD). The objective of this project was to test the immunogenicity of the fusion protein PAD and to compare its protective activity against S. pneumoniae infection with PA or PD alone and a combination of PA and PD. The prediction of physicochemical properties, antigenicity, allergenicity, toxicity, and 3D-structure of the constructs, as well as molecular docking with HLA receptor and immune simulation were performed using computational tools. Finally, mice were immunized and the serum levels of antibodies/cytokines and functionality of antibodies in vitro were evaluated after immunization. The mice survival rates and decrease of bacterial loads in the blood/spleen were examined following the challenge.ResultsThe computational analyses indicated the proposed constructs could be antigenic, non-allergenic, non-toxic, soluble and able to elicit robust immune responses. The results of actual animal experiments revealed the candidate vaccines could induce the mice to produce high levels of antibodies and cytokines. The complement-mediated bactericidal activity of antibodies was confirmed and the antibodies provided favorable survival in immunized mice after bacterial challenge. In general, the experimental results verified the immunoinformatics studies.ConclusionFor the first time this report presents novel peptide-based vaccine candidates consisting of immunodominant regions of PspA and PhtD antigens. The obtained findings confirmed that the fusion formulation could be relatively more efficient than the individual and combination formulations. The results propose that the fusion protein alone could be used as a serotype-independent pneumococcal vaccine or as an effective partner protein for a conjugate polysaccharide vaccine.

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New insights into histidine triad proteins: solution structure of a Streptococcus pneumoniae PhtD domain and zinc transfer to AdcAII.

Publications

New insights into histidine triad proteins: solution structure of a Streptococcus pneumoniae PhtD domain and zinc transfer to AdcAII.

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