Knejski Paweł P, Erramilli Satchal K, Kossiakoff Anthony A
Deparment of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
Laboratory of Medical Biology, Faculty of Biotechnology, University of Wrocław, Wrocław 50-383, Poland.
bioRxiv. 2024 Aug 5:2023.08.01.551527. doi: 10.1101/2023.08.01.551527.
Pathogenic bacteria, such as , depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake protein in clinical isolates. However, a comprehensive understanding of heme recognition and TBDT transport mechanisms, especially PhuR, remains limited. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) and a phage display-generated synthetic antibody (sAB) as a fiducial marker to enable the determination of a high-resolution (2.5 Å) structure of PhuR with a bound heme. Notably, the structure reveals iron coordination by Y529 on a conserved extracellular loop, shedding light on the role of tyrosine in heme binding. Biochemical assays and negative-stain EM demonstrated that the sAB specifically targets the heme-bound state of PhuR. These findings provide insights into PhuR's heme binding and offer a template for developing conformation-specific sABs against outer membrane proteins (OMPs) for structure-function investigations.
致病性细菌，如 ，依靠清除血红素来获取铁这一必需营养素。依赖TonB的转运蛋白（TBDT）PhuR是临床分离株中主要的血红素摄取蛋白。然而，对血红素识别和TBDT转运机制，尤其是PhuR的全面理解仍然有限。在本研究中，我们采用单颗粒低温电子显微镜（cryo-EM）和噬菌体展示产生的合成抗体（sAB）作为基准标记，以确定结合血红素的PhuR的高分辨率（2.5 Å）结构。值得注意的是，该结构揭示了保守细胞外环上的Y529对铁的配位作用，阐明了酪氨酸在血红素结合中的作用。生化分析和负染EM表明，sAB特异性靶向PhuR的血红素结合状态。这些发现为PhuR的血红素结合提供了见解，并为开发针对外膜蛋白（OMP）的构象特异性sAB以进行结构功能研究提供了模板。
