Montserrat-Canals Mateu, Bjerregaard-Andersen Kaare, Sørensen Henrik Vinther, Kommedal Eirik, Cordara Gabriele, Vaaje-Kolstad Gustav, Krengel Ute
Centre for Molecular Medicine Norway, University of Oslo, NO-0318 Oslo, Norway.
Department of Chemistry, University of Oslo, NO-0315 Oslo, Norway.
QRB Discov. 2024 Dec 26;5:e12. doi: 10.1017/qrd.2024.14. eCollection 2024.
Despite major efforts toward its eradication, cholera remains a major health threat and economic burden in many low- and middle-income countries. Between outbreaks, the bacterium responsible for the disease, , survives in aquatic environmental reservoirs, where it commonly forms biofilms, for example, on zooplankton. -acetyl glucosamine-binding protein A (GbpA) is an adhesin that binds to the chitinaceous surface of zooplankton and breaks its dense crystalline packing thanks to its lytic polysaccharide monooxygenase (LPMO) activity, which provides with nutrients. In addition, GbpA is an important colonization factor associated with bacterial pathogenicity, allowing the binding to mucins in the host intestine. Here, we report the discovery of a cation-binding site in proximity of the GbpA active site, which allows Ca, Mg, or K binding close to its carbohydrate-binding surface. In addition to the X-ray crystal structures of cation-LPMO complexes (to 1.5 Å resolution), we explored how the presence of ions affects the stability and activity of the protein. Calcium and magnesium ions were found to bind to GbpA specifically, with calcium ions - abundant in natural sources of chitin - having the strongest effect on protein stability. When the cation-binding site was rendered non-functional, a decrease in activity was observed, highlighting the importance of the structural elements stabilized by calcium. Our findings suggest a cation-binding site specific to GbpA and related LPMOs that may fine-tune binding and activity for its substrates during environmental survival and host infection.
尽管为根除霍乱做出了重大努力，但霍乱在许多低收入和中等收入国家仍然是主要的健康威胁和经济负担。在疫情爆发期间，导致该疾病的细菌在水生环境蓄水池中存活，在那里它通常形成生物膜，例如在浮游动物上。N-乙酰葡糖胺结合蛋白A（GbpA）是一种粘附素，它与浮游动物的几丁质表面结合，并由于其裂解多糖单加氧酶（LPMO）活性而破坏其密集的晶体堆积，该活性为细菌提供营养。此外，GbpA是一种与细菌致病性相关的重要定植因子，可使其与宿主肠道中的粘蛋白结合。在此，我们报告在GbpA活性位点附近发现了一个阳离子结合位点，该位点允许Ca、Mg或K在其碳水化合物结合表面附近结合。除了阳离子-LPMO复合物的X射线晶体结构（分辨率达到1.5 Å）外，我们还探究了离子的存在如何影响蛋白质的稳定性和活性。发现钙离子和镁离子特异性地与GbpA结合，在天然几丁质来源中含量丰富的钙离子对蛋白质稳定性的影响最强。当阳离子结合位点失去功能时，观察到活性降低，这突出了由钙稳定的结构元件的重要性。我们的研究结果表明，GbpA和相关LPMO具有一个特定的阳离子结合位点，该位点可能在环境生存和宿主感染期间微调其对底物的结合和活性。
