Zhu Wen, Iavarone Anthony T, Klinman Judith P
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, United States.
California Institute for Quantitative Biosciences, University of California, Berkeley, California 94720, United States.
ACS Cent Sci. 2024 Jan 17;10(2):251-263. doi: 10.1021/acscentsci.3c01023. eCollection 2024 Feb 28.
Interactions among proteins and peptides are essential for many biological activities including the tailoring of peptide substrates to produce natural products. The first step in the production of the bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS) to characterize the structure and conformational dynamics in the protein-protein and protein-peptide complexes necessary for PqqE function. HDX-MS-identified hotspots can be discerned in binary and ternary complex structures composed of the peptide PqqA, the peptide-binding chaperone PqqD, and PqqE. Structural conclusions are supported by size-exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS). HDX-MS further identifies reciprocal changes upon the binding of substrate peptide and S-adenosylmethionine (SAM) to the PqqE/PqqD complex: long-range conformational alterations have been detected upon the formation of a quaternary complex composed of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA binding site in PqqD to the PqqE active site FeS. Interactions among the various regions are concluded to arise from both direct contact and distal communication. The described experimental approach can be readily applied to the investigation of protein conformational communication among a large family of peptide-modifying rSAM enzymes.
蛋白质与肽之间的相互作用对于许多生物活性至关重要，包括对肽底物进行修饰以产生天然产物。从其肽前体生产细菌氧化还原辅因子吡咯并喹啉醌（PQQ）的第一步由一种自由基S-腺苷甲硫氨酸（rSAM）酶PqqE催化。我们描述了如何使用氢-氘交换质谱（HDX-MS）来表征PqqE功能所必需的蛋白质-蛋白质和蛋白质-肽复合物中的结构和构象动力学。在由肽PqqA、肽结合伴侣PqqD和PqqE组成的二元和三元复合物结构中，可以识别出HDX-MS鉴定的热点。结构结论得到了尺寸排阻色谱与小角X射线散射联用（SEC-SAXS）的支持。HDX-MS进一步确定了底物肽和S-腺苷甲硫氨酸（SAM）与PqqE/PqqD复合物结合后的相互变化：在由PqqA/PqqD/PqqE和SAM组成的四元复合物形成时，检测到了远程构象改变，跨度近40 Å，从PqqD中的PqqA结合位点到PqqE活性位点FeS。得出结论，各个区域之间的相互作用源于直接接触和远程通讯。所描述的实验方法可以很容易地应用于研究一大类肽修饰rSAM酶之间的蛋白质构象通讯。
