Domain Flexibility in Ligand-Free and Inhibitor-BoundEscherichia coliAdenylate Kinase Based on a Mode-Coupling Analysis of¹⁵N Spin Relaxation

Abstract

Adenylate kinase from Escherichia coli (AKeco), consisting of a 23.6-kDa polypeptide chain folded into domains CORE, AMPbd, and LID catalyzes the reaction AMP + ATP T 2ADP. The domains AMPbd and LID execute large-amplitude movements during catalysis. Backbone dynamics of ligandfree and AP 5 A-inhibitor-bound AKeco is studied with slowly relaxing local structure (SRLS) 15 N relaxation, an approach particularly suited when the global (τ m ) and the local (τ) motions are likely to be coupled. For AKeco τ m ) 15.1 ns, whereas for AKeco*AP 5 A τ m ) 11.6 ns. The CORE domain of AKeco features an average squared order parameter, <S 2 >, of 0.84 and correlation times τ f ) 5-130 ps. Most of the AKeco*AP 5 A backbone features <S 2 > ) 0.90 and τ f ) 33-193 ps. These data are indicative of relative rigidity. Domains AMPbd and LID of AKeco, and loops 1 /R 1 , R 2 /R 3 , R 4 / 3 , R 5 / 4 , and 8 /R 7 of AKeco*AP 5 A, feature a novel type of protein flexibility consisting of nanosecond peptide plane reorientation about the C i-1 R -C i R axis, with correlation time τ ⊥ ) 5.6-11.3 ns. The other microdynamic parameters underlying this dynamic model include S 2 ) 0.13-0.5, τ || on the ps time scale, and a diffusion tilt MD ranging from 12 to 21°. For the ligand-free enzyme the τ ⊥ mode was shown to represent segmental domain motion, accompanied by conformational exchange contributions R ex e 4.4 s -1 . Loop R 4 / 3 and R 5 / 4 dynamics in AKeco*AP 5 A is related to the "energetic counter-balancing of substrate binding" effect apparently driving kinase catalysis. The other flexible AKeco*AP 5 A loops may relate to domain motion toward product release.The ability to interpret nuclear spin relaxation properties in terms of microdynamic parameters turned NMR into a powerful method for elucidating protein dynamics (1, 2). The amide 15 N spin in proteins is a particularly useful probe, relaxed predominantly by dipolar coupling to the amide proton and 15 N chemical shift anisotropy (CSA) 1 (3). The experimental NMR observables are controlled by the global and local dynamic processes experienced by protein N-H bond vectors, which determine the spectral density function, J(ω). 15 N relaxation data in proteins are commonly analyzed with the model-free (MF) approach, where the global and local motions are assumed to be decoupled (4-6). In a recent study (7), we applied the two-body slowly relaxing local structure (SRLS) approach developed by Freed and coworkers (8, 9) to 15 N relaxation in proteins. SRLS accounts rigorously for dynamical coupling between the local and global motions, and treats the global diffusion, the local diffusion, the local ordering, and the magnetic interactions as tensors that may be tilted relative to one another, providing thereby important information related to protein structure (10-12). The MF spectral density functions constitute asymptotic solutions of the SRLS spectral densities (7,8,13). It was found that currently available experimental 15 N relaxation data are sensitive to the coupling-induce...

A Model of Interdomain Mobility in a Multidomain Protein

“…The parameters of the ITS model were obtained by minimizing the following target function: (16) where for each of the N residues included in the sum, is the value of ρ derived directly from the experimental data according to the first equality in Eq. 13 and is obtained by substituting into the second equality in Eq.…”

An Improved Picture of Methyl Dynamics in Proteins from Slowly Relaxing Local Structure Analysis of 2H Spin Relaxation

“…[16][17][18][19][20][21][22][23] Two rotators, representing the global motion of the protein, R C , and the local motion of the probe (C-D bond in this case), R L , are treated. The motions of the protein and the probe are coupled by a local potential, U(Ω C'M ), where C' denotes the local director fixed in the protein, and M the local ordering/local diffusion frame fixed in the probe.…”

“…[16][17][18][19][20][21][22][23] This has been accomplished by applying to NMR spin relaxation in proteins 16 the Slowly Relaxing Local Structure (SRLS) approach of Freed and co-workers. [24][25][26] SRLS can be considered the generalization of MF, yielding the latter in asymptotic limits.…”

“…It features explicitly local motional modes parallel and perpendicular to the methyl rotation axis. [16][17][18][19][20][21][22][23]25,26 and accounts rigorously for the tilt between a rhombic local ordering frame, M, and the magnetic frame, Q. The Euler angles Ω C'M , which relate the M frame to a local director frame, C' (e.g., the equilibrium C-CH 3 orientation), are governed by the coupling/ ordering potential.…”

On the measurement of 15N–{1H} nuclear Overhauser effects

“…Nuclear Magnetic Resonance (NMR) is a powerful tool to study protein dynamics over a wide range of timescales [1,2]. Hydrodynamic properties of proteins in solution and dynamics occurring faster than a few nanoseconds are usually measured by the quantitative analysis of 15 N relaxation rates [2][3][4][5]. These rates depend both on the overall rotational diffusion in solution as well as fast backbone motions on a subnanosecond timescale through their effects on the spectral density functions [6][7][8][9].…”