Cryptic Binding Pockets in PDC‑3 β‑Lactamase Modulate Resistance Profiles

Cryptic binding pockets
in proteins can modulate catalysis,
allostery,
and druggability. Yet they are rarely captured by experiments or conventional
molecular dynamics simulations. Here, we combine enhanced sampling
with an unsupervised deep-learning pipeline to map the full conformational
landscape of the Ω-loop in class C β-lactamase PDC-3.
Three principal conformational ensembles were identified: a crystal-like
state resembling the native structure, an expansive state characterized
by widening of the active-site cleft, and a constricted state that
blocks access to the catalytic site. Residues 219 and 221 act as molecular
switches that shuffle the enzyme between these states and thereby
modulate the resistance profiles. Steady-state inhibition assays with
nitrocefin and bulky cephalosporins confirm that substitutions at
these positions selectively reshaped the binding pocket. In addition,
across multiple expansive states, D217 repeatedly forms a salt bridge
with K67 in a geometry reminiscent of general base E166 of class
A enzymes. Thus, it is plausible that D217 might transiently adopt
a ‘backup’ general base role under certain conformational
states. Most strikingly, occlusion of the catalytic site reveals a
previously unseen cryptic pocket, offering an attractive allosteric
target for inhibitors that would lock PDC-3 in a catalytically incompetent
conformation. The integrated framework proposed in this study is highly
generalizable and can serve as a powerful tool for identifying hidden
protein conformations and uncovering previously inaccessible regulatory
mechanisms.

蛋白质中的隐蔽结合口袋（Cryptic binding pockets）可调控催化作用、别构效应与成药性。然而这类口袋极少能通过实验或常规分子动力学模拟被捕获。本研究将增强采样（enhanced sampling）技术与无监督深度学习（unsupervised deep-learning）流程相结合，对C类β-内酰胺酶PDC-3的Ω环（Ω-loop）的完整构象景观进行了图谱绘制。本研究共鉴定出三种主要构象集合（principal conformational ensembles）：一是类晶体构象，与天然结构高度相似；二是以活性位点裂隙（active-site cleft）扩张为特征的扩张态；三是阻塞催化位点（catalytic site）入口的收缩态。残基219与221充当分子开关（molecular switches），介导酶在这三种构象间相互切换，进而调控其耐药谱（resistance profiles）。针对硝噻吩（nitrocefin）与大体积头孢菌素的稳态抑制实验（steady-state inhibition assays）证实，对上述位点的氨基酸取代可选择性重塑结合口袋。此外，在多种扩张态构象中，残基D217会反复与残基K67形成盐桥（salt bridge），其几何构型与A类酶的广义碱（general base）E166类似。由此推测，D217或可在特定构象状态下临时充当‘后备’广义碱。最引人注目的是，催化位点的阻塞暴露出一个此前未被发现的隐蔽口袋，为开发能将PDC-3锁定为无催化活性构象（catalytically incompetent conformation）的抑制剂提供了极具潜力的变构靶点（allosteric target）。本研究提出的整合框架（integrated framework）具有极强的泛用性，可作为识别隐藏蛋白质构象、发掘此前未被探明的调控机制的有力工具。