Putative copper‐ and zinc‐binding motifs in Streptococcus pneumoniae identified by immobilized metal affinity chromatography and mass spectrometry

Abstract

The aim of metalloproteomics is to identify and characterize putative metal-binding proteins and metal-binding motifs. In this study, we performed a systematical metalloproteomic analysis on Streptococcus pneumoniae through the combined use of efficient immobilized metal affinity chromatography enrichment and high-accuracy linear ion trap-Orbitrap MS to identify metal-binding proteins and metal-binding peptides. In total, 232 and 166 putative metal-binding proteins were respectively isolated by Cu- and Zn-immobilized metal affinity chromatography columns, in which 133 proteins were present in both preparations. The putative metalloproteins are mainly involved in protein, nucleotide and carbon metabolisms, oxidation and cell cycle regulation. Based on the sequence of the putative Cu- and Zn-binding peptides, putative Cu-binding motifs were identified: H(X)mH (m=0-11), C(X)(2) C, C(X)nH (n=2-4, 6, 9), H(X)iM (i=0-10) and M(X)tM (t=8 or 12), while putative Zn-binding motifs were identified as follows: H(X)mH (m=1-12), H(X)iM (i=0-12), M(X)tM (t=0, 3 and 4), C(X)nH (n=1, 2, 7, 10 and 11). Equilibrium dialysis and inductively coupled plasma-MS experiments confirmed that the artificially synthesized peptides harboring differential identified metal-binding motifs interacted directly with the metal ions. The metalloproteomic study presented here suggests that the comparably large size and diverse functions of the S. pneumoniae metalloproteome may play important roles in various biological processes and thus contribute to the bacterial pathologies.

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

“…In bacteria, several proteins involved in cell division, cell wall synthesis, and remodeling, including peptidoglycan deacetylases and metalloproteases, use zinc as a cofactor (21,44). We examined whether the absence of the Lmb/AdcA/AdcAII zincbinding proteins or the AdcCB translocon affected cell morphology and/or chain formation.…”

“…Zinc is a cofactor for a number of prokaryotic enzymes. In bacteria, there are several important enzymes that are zinc-containing proteins, including alcohol dehydrogenase, peptidoglycan deacetylase, metalloprotease, and ribosomal proteins (21,44). Homologs of these proteins are present in S. agalactiae and are also likely to require zinc for their proper activity.…”

The roles of transition metals in the physiology and pathogenesis of Streptococcus pneumoniae

“…Accordingly, copper negatively affects iron-sulfur clusters of dehydratases, inhibits branched chain amino acid biosynthesis, photosystem oxidase, and displaces manganese in SOD as additional mechanisms of toxicity (Batinic-Haberle et al, 1997; Macomber and Imlay, 2009; Azzouzi et al, 2013). Ribonucleotide reductase protein, NrdF, and coupled factor NrdE are both necessary for the aerobic nucleotide synthesis pathway in pneumococci and may interact with copper, as may other proteins involved in aerobic nucleotide synthesis, such as NrdI (Sun et al, 2011). Thus, it is also possible that copper is causing toxicity by inhibiting nucleotide synthesis or other essential metabolic processes.…”

Microbial Metalloproteomics

“…One of particular interest for metalloproteomics is the use of IMAC (Immobilized Metal-ion Affinity Chromatography) to enrich the sample with proteins that are able to bind particular metal ions. This has been used previously to find Cu 2+ , Zn 2+ , Ni 2+ and Co 2+ binding proteins in Streptococcus pneumonia and Ni 2+ and Bi 2+ in Helicobacter pylori [25,26,27,28]. Recently, magnetic microspheres have been developed to perform IMAC [29].…”