The Active Site of a Prototypical “Rigid” Drug Target is Marked by Extensive Conformational Dynamics

Abstract

Drug discovery, in particular optimization of candidates using medicinal chemistry, is generally guided by structural biology. However, for optimizing binding kinetics, relevant for efficacy and off‐target effects, information on protein motion is important. Herein, we demonstrate for the prototypical textbook example of an allegedly “rigid protein” that substantial active‐site dynamics have generally remained unrecognized, despite thousands of medicinal‐chemistry studies on this model over decades. Comparing cryogenic X‐ray structures, solid‐state NMR on micro‐crystalline protein at room temperature, and solution NMR structure and dynamics, supported by MD simulations, we show that under physiologically relevant conditions the pocket is in fact shaped by pronounced open/close conformational‐exchange dynamics. The study, which is of general significance for pharmacological research, evinces a generic pitfall in drug discovery routines.

Allostery at a protein-protein interface harboring an intermolecular dynamic network

“…The experimental demonstration of intermolecular protein-protein interactions that induce coupling of μs timescale motion between residues of the same protein sheds light on how biological interaction partners can allosterically influence protein functionality without firm, visible changes in ground-state structures. In particular, the experiment demonstrates why allosteric modulation cannot only modulate target function destructively due to disruption of local dynamics or of dynamic networks, e. g., through steric blockage or rigidification of protein segments, as associated with some orthosteric inhibitors 25,26 . Instead, allosteric modulation seems possible in a constructive way, by adding missing links between individual residues or into partially preformed networks.…”

Protein structural dynamics by Magic-Angle Spinning NMR

“…Moreover, a coupling was observed between motions of side chains coordinating the copper in the active site and those of distant backbone elements of the protein which are pivotal for SOD stability and efficient enzymatic activity [33]. The multitimescale dynamics unveiled in these studies by solid-state NMR are particularly difficult to observe using other methods [34], and in the specific case of SOD had escaped previous determinations by solution NMR.…”

Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR

“…Unlike solution-state NMR, magic-angle spinning (MAS) NMR does not face inherent protein size limitations and allows to see, in principle, each atom. MAS NMR is a powerful technique for studying dynamics at atomic resolution, and has been applied to sedimented, crystalline, membrane and amyloid proteins (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47). Most of the previous MAS NMR dynamics studies focused on proteins below 20-30 kDa, as the resonance overlap often encountered in larger proteins complicates analyses.…”