New Insights into Histidine Triad Proteins: Solution Structure of a Streptococcus pneumoniae PhtD Domain and Zinc Transfer to AdcAII

Abstract

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.

The Adc/Lmb System Mediates Zinc Acquisition in Streptococcus agalactiae and Contributes to Bacterial Growth and Survival

“…The histidine-rich loop and ZinT domain of the S. pneumoniae AdcA homolog have been suggested to aid in recruiting Zn 2ϩ (40). The structural differences between S. agalactiae AdcA and Lmb/ AdcAII proteins are similar to those observed between S. pneumoniae AdcA and AdcAII proteins and correspond to distinct zinc acquisition mechanisms (40,41).…”

“…These genes encode polyhistidine triad proteins, and S. agalactiae Sht has been shown to promote complement degradation by binding to factor H (12). In S. pneumoniae, the Sht homologs, called phtproteins, aid in zinc delivery to the ABC transporter substrate-binding protein AdcAII (40,41) and play a role in pneumococcal adhesion to the respiratory epithelium (57). The involvement of S. agalactiae Sht proteins in zinc transport, their interaction with the Adc/Lmb system components, and their putative redundancy are now under study and should provide new insights to better understand mechanisms of zinc acquisition in S. agalactiae.…”

Overlapping Functionality of the Pht Proteins in Zinc Homeostasis of Streptococcus pneumoniae

“…However, it is also plausible that they could help defend the pneumococcus when an excess of the metal ion is present in the extracellular environment, which exerts a toxic effect on the bacteria by interfering with the physiological function of the ABC permease, PsaBCA, in importing the essential transition metal ion manganese (20,21). It has been speculated that the Pht proteins may aid in protecting the PsaBCA permease from extracellular Zn 2ϩ by binding excess ions and acting as a "sink" (12,22). To test this hypothesis, wild-type and ⌬phtABDE pneumococci were first grown in CDM-TPEN to induce maximal expression of pht genes (6, 9) and then subcultured into CDM supplemented with increasing concentrations of ZnSO 4 (Fig.…”

“…Zinc acquisition is critical for the survival of S. pneumoniae during infection and occurs via the ATP-binding cassette (ABC) transporter AdcBC after delivery of the metal via the substrate-binding proteins (SBPs) AdcA and/or AdcAII (7)(8)(9)(10)(11). There is now substantial evidence supporting the hypothesis that an important role of the Pht proteins is to facilitate Zn 2ϩ acquisition via AdcAII (6,9,12). The phtD and adcAII genes are cotranscribed, and their expression is coregulated by cellular Zn 2ϩ abundance.…”

“…The phtD and adcAII genes are cotranscribed, and their expression is coregulated by cellular Zn 2ϩ abundance. The two proteins have been shown to interact with one another by in vitro cross-linking and immunoprecipitation experiments (6,12). Recently we showed that the growth of a ⌬phtABDE ⌬adcA mutant was highly attenuated in media containing a low concentration of available Zn 2ϩ compared to the growth of the ⌬adcA strain, and the in vivo fitness of a ⌬phtABDE ⌬adcA mutant was significantly less than that of the ⌬phtABDE ⌬adcAII strain (9).…”