Abstract
Streptococcus pneumoniae scavenges essential zinc ions from the host during colonization and infection. This is achieved by the ATP-binding cassette transporter, AdcCB, and two solute-binding proteins (SBPs), AdcA and AdcAII. It has been established that AdcAII serves a greater role during initial infection, but the molecular details of how the protein selectively acquires Zn(II) remain poorly understood. This can be attributed to the refractory nature of metal-free AdcAII to high-resolution structural determination techniques. Here, we overcome this issue by separately mutating the Zn(II)-coordinating residues and performing a combination of structural and biochemical analyses on the variant proteins. Structural analyses of Zn(II)-bound AdcAII variants revealed that specific regions within the protein underwent conformational changes via direct coupling to each of the metal-binding residues. Quantitative in vitro metal-binding assays combined with affinity determination and phenotypic growth assays revealed that each of the four Zn(II)-coordinating residues contributes to metal binding by AdcAII. Intriguingly, the phenotypic growth impact of the mutant adcAII alleles was, in general, independent of affinity, suggesting that the Zn(II)-bound conformation of the SBP is crucial for efficacious metal uptake. Collectively, these data highlight the intimate coupling of ligand affinity with protein conformational change in ligand-receptor proteins and provide a putative mechanism for AdcAII. These findings provide further mechanistic insight into the structural and functional diversity of SBPs that is broadly applicable to other prokaryotes.
Highlights
Zinc is an essential micronutrient in all domains of life
These data suggest that mutations that perturb the Zn(II)-coordinating site or alter global protein structure are poorly tolerated in AdcAII
S. pneumoniae survival is dependent on the efficacious acquisition of Zn(II) from the host during infection (Bayle et al, 2011; Plumptre et al, 2014), with prior studies establishing the crucial role of AdcAII in initial infection and invasive disease (Plumptre et al, 2014; Brown et al, 2016)
Summary
Zinc is an essential micronutrient in all domains of life. Zinc, which occurs as the divalent cation Zn(II), fulfils both structural and functional roles within proteins, and is estimated to be required by up to 6% of the bacterial proteome (Andreini et al, 2006). To overcome host-imposed Zn(II) restriction, pathogenic bacteria have evolved high-affinity acquisition systems to compete for metal ions at the hostpathogen interface (Ammendola et al, 2007; Sabri et al, 2009; Bayle et al, 2011; Pederick et al, 2015) These include the ubiquitous Type II ATP-binding cassette (ABC) family importers (Lewis et al, 2012), zinc-iron permease (ZIP) transporters (Grass et al, 2005; Karlinsey et al, 2010), P-type ATPases (Lewinson et al, 2009; Chien et al, 2013), and the recently identified zincophore scavenging systems (Lhospice et al, 2017; Morey and Kehl-Fie, 2020). Bacterial ABC permeases employ extra-cytoplasmic solute-binding proteins (SBPs) to obtain cargo such as Zn(II) ions from the periplasm (Gram-negative bacteria) or the extracellular environment (Grampositive bacteria) (Lewis et al, 2012)
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