A Trap-Door Mechanism for Zinc Acquisition by Streptococcus pneumoniae AdcA

Abstract

Zinc is an essential element in all domains of life. Nonetheless, how prokaryotes achieve selective acquisition of zinc from the extracellular environment remains poorly understood. Here, we elucidate a novel mechanism for zinc-binding in AdcA, a solute-binding protein of Streptococcus pneumoniae. Crystal structure analyses reveal the two-domain organization of the protein and show that only the N-terminal domain (AdcAN) is necessary for zinc import. Zinc binding induces only minor changes in the global protein conformation of AdcA and stabilizes a highly mobile loop within the AdcAN domain. This loop region, which is conserved in zinc-specific solute-binding proteins, facilitates closure of the AdcAN binding site and is crucial for zinc acquisition. Collectively, these findings elucidate the structural and functional basis of selective zinc uptake in prokaryotes. IMPORTANCE Zinc is an essential nutrient for the virulence of bacterial pathogens such as Streptococcus pneumoniae. Many Gram-positive bacteria use a two-domain lipoprotein for zinc acquisition, but how this class of metal-recruiting proteins acquire zinc and interact with the uptake machinery has remained poorly defined. We report the first structure of a two-domain lipoprotein, AdcA from S. pneumoniae, and use computational, spectroscopic, and microbiological approaches to provide new insights into the functional basis of zinc recruitment. Our findings reveal that AdcA employs a novel mechanism for zinc binding that we have termed the “trap-door” mechanism, and we show how the static metal-binding site of the protein, which confers its selectivity for zinc ions, is combined with a dynamic surface element to facilitate zinc recruitment and import into the bacterium. Together, these findings expand our understanding of how bacteria acquire zinc from the environment and provide a foundation for inhibiting this process, through antimicrobial targeting of the dynamic structural elements to block bacterial zinc scavenging.

Conformation of the Solute-Binding Protein AdcAII Influences Zinc Uptake in Streptococcus pneumoniae

“…The interaction of His65 with Zn(II) facilitates movement of loop α2β2 onto the binding site. Interestingly, although loop α2β2 is mobile in AdcAII, it lacks the extensive mobility observed for the comparable loop in AdcA ( Luo et al., 2021 ). This highlights that loop α2β2 contributes differently to the Zn(II) recruitment process in the two SBPs, despite their interaction with the same transporter.…”

“…For this, the Zn(II)-coordinating residues were substituted with an uncharged residue. Alanine was substituted for His65, which has recently been implicated as a component of a crucial mobile loop in S. pneumoniae AdcA ( Luo et al., 2021 ), and His141. Leucine and glutamine were substituted for residues His205 and Glu280, respectively, to preserve steric bulk.…”

“…When considered in context with the Zn-AdcAII H65A structure, these data suggest that loop α2β2 controls access to the N-terminal side of the metal-binding site, with its conformation being restricted by interaction of His65 with the Zn(II) ion. Intriguingly, the loop α2β2 region was recently shown to be critical for the function of AdcA for S. pneumoniae Zn(II) uptake in a Δ adcAII background ( Luo et al., 2021 ). However, the high mobility and dynamic conformational sampling of loop α2β2 in the metal-free state of AdcA is distinctly different from the equivalent region in AdcAII ( Figure 3 ).…”

“…The structural rearrangements in the C-terminal lobe of AdcAII have been observed in E. coli ZnuA (PDB: 2PS0 [Zn(II)-bound]; 2PS3 [metal-free]), suggesting that β-sheet formation and subsequent rotation of the α-helices are common features of Zn(II) SBPs ( Yatsunyk et al., 2008 ). Structural studies of ZnuA, as well as P. denitrificans AztC (PDB: 5W57 and 5W56), Candidatus Liberibacter asiaticus ZnuA2 (PDB: 4UDN and 4UDO) and S. pneumoniae AdcA (PDB: 7JJ9 [AdcA] and 7JJ8 [N-terminal AdcA domain]) also show that the N-terminal lobe binding site His residue and the loop in which it resides, His65 and loop α2β2 in AdcAII, are commonly engaged in structural rearrangements upon Zn(II)-binding ( Yatsunyk et al., 2008 ; Sharma et al., 2015 ; Neupane et al., 2017 ; Luo et al., 2021 ). However, our study reveals that although the Zn(II)-induced conformational changes are relatively subtle, their concerted action is required for high ligand affinity and optimal uptake of Zn(II).…”

“…Although both AdcA and AdcAII share this conserved fold, AdcA contains additional structural elements associated with Zn(II) recruitment ( Plumptre et al., 2014 ). These are an extended C-terminal domain, which has homology to the Gram-negative Zn(II) chaperone ZinT, and a histidine-rich loop, which is common in Zn(II)-specific SBPs ( Plumptre et al., 2014 ; Luo et al., 2021 ). AdcAII lacks these regions and although it does have a flexible loop of residues near the metal binding site, this loop is devoid of histidine residues ( Wei et al., 2007 ; Loisel et al., 2008 ; Petrarca et al., 2010 ).…”