Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, Australia.
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia.
Front Cell Infect Microbiol. 2021 Aug 19;11:729981. doi: 10.3389/fcimb.2021.729981. eCollection 2021.
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 direct coupling to each of the metal-binding residues. Quantitative 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 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.
在定植和感染过程中，它从宿主中掠夺必需的锌离子。这是通过 ATP 结合盒转运蛋白 AdcCB 和两个溶质结合蛋白（SBP）AdcA 和 AdcAII 实现的。已经确定，AdcAII 在初始感染中起更大的作用，但蛋白质如何选择性地获取 Zn(II)的分子细节仍知之甚少。这可以归因于无金属 AdcAII 对高分辨率结构测定技术的难治性。在这里，我们通过分别突变锌（II）配位残基并对变体蛋白进行结构和生化分析的组合来克服这个问题。锌（II）结合的 AdcAII 变体的结构分析表明，蛋白质内的特定区域发生了构象变化，直接与每个金属结合残基偶联。定量金属结合测定与亲和力测定和表型生长测定相结合，表明 AdcAII 的四个锌（II）配位残基中的每一个都有助于金属结合。有趣的是，突变等位基因的表型生长影响通常与亲和力无关，这表明 SBP 的锌（II）结合构象对于有效的金属摄取至关重要。总的来说，这些数据突出了配体亲和力与配体受体蛋白中蛋白质构象变化的紧密偶联，并为 AdcAII 提供了一个可能的机制。这些发现为 SBP 的结构和功能多样性提供了进一步的机制见解，这在其他原核生物中具有广泛的适用性。
