Hydrogen–Deuterium Exchange Mass Spectrometry Identifies Local and Long-Distance Interactions within the Multicomponent Radical SAM Enzyme, PqqE

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 Fe4S4. 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是诸多生物学过程的核心基础，其中涵盖了对肽底物进行定制化修饰以合成天然产物的步骤。从肽前体合成细菌来源的氧化还原辅酶吡咯并喹啉醌（pyrroloquinoline quinone, PQQ）的首步反应，由自由基SAM（radical SAM, rSAM）酶PqqE催化完成。本研究采用氢-氘交换质谱（hydrogen–deuterium exchange mass spectrometry, HDX-MS）技术，对PqqE行使功能所必需的蛋白质-蛋白质及蛋白质-肽复合物的结构与构象动态进行表征。在由肽PqqA、肽结合伴侣蛋白PqqD与PqqE组成的二元及三元复合物结构中，可识别出HDX-MS鉴定得到的相互作用热点区域。尺寸排阻色谱-小角X射线散射联用（size-exclusion chromatography coupled to small-angle X-ray scattering, SEC-SAXS）的实验数据佐证了上述结构推断。HDX-MS还揭示了底物肽与S-腺苷甲硫氨酸（S-adenosylmethionine, SAM）结合至PqqE/PqqD复合物后产生的双向构象变化：当由PqqA、PqqD、PqqE与SAM组成的四元复合物形成时，可检测到跨度近40埃的长距离构象改变，该变化覆盖了从PqqD内的PqqA结合位点到PqqE活性位点Fe4S4簇的区域。研究推断，各区域间的相互作用既源于直接接触，也涉及远端信号通讯。本研究所述的实验方法可直接推广应用于探究一大类肽修饰型rSAM酶之间的蛋白质构象通讯机制。