A Quantitative Model for cAMP Binding to the Binding Domain of MloK1

Abstract

Ligand-protein binding processes are essential in biological systems. A well-studied system is the binding of cyclic adenosine monophosphate to the cyclic nucleotide binding domain of the bacterial potassium channel MloK1. Strikingly, the measured on-rate for cyclic adenosine monophosphate binding is two orders of magnitude slower than a simple Smoluchowski diffusion model would suggest. To resolve this discrepancy and to characterize the ligand-binding path in structural and energetic terms, we calculated 1100 ligand-binding molecular dynamics trajectories and tested two scenarios: In the first scenario, the ligand transiently binds to the protein surface and then diffuses along the surface into the binding site. In the second scenario, only ligands that reach the protein surface in the vicinity of the binding site proceed into the binding site. Here, a binding funnel, which increasingly confines the translational as well as the rotational degrees of freedom, determines the binding pathways and limits the on-rate. From the simulations, we identified five surface binding states and calculated the rates between these surface binding states, the binding site, and the bulk. We find that the transient binding of the ligands to the surface binding states does not affect the on-rate, such that this effect alone cannot explain the observed low on-rate. Rather, by quantifying the translational and rotational degrees of freedom and by calculating the binding committor, our simulations confirmed the existence of a binding funnel as the main bottleneck. Direct binding via the binding funnel dominates the binding kinetics, and only ∼10% of all ligands proceed via the surface into the binding site. The simulations further predict an on-rate between 15 and 40μs(mol/l), which agrees with the measured on-rate.

Thermodynamics of camphor migration in cytochrome P450cam by atomistic simulations

“…Second, the process is not at equilibrium. To sample ligand diffusion within timescales accessible in MD simulations computing nonequilibrium trajectories is necessary since ligand binding occurs on the microsecond-millisecond time scale 12 . Thus, the ligand diffusion and its escape rates are accelerated, making it difficult to estimate thermodynamic and kinetic properties from the simulations.…”

Heterogeneous and rate-dependent streptavidin–biotin unbinding revealed by high-speed force spectroscopy and atomistic simulations

“…At lower loading rates, states Int 3 and Int 4, farther out, are also visited. Likely these, and even intermediates lying farther outside, provide a rugged funnel (41–43) for rebinding under equilibrium conditions.…”

Spatiotemporal Resolution of Conformational Changes in Biomolecules by Combining Pulsed Electron–Electron Double Resonance Spectroscopy with Microsecond Freeze-Hyperquenching

“…This movement has been inferred from X-ray 49 and NMR 50,51 structures of the CNBD in its apo and holo states and has also been predicted by atomistic simulations. 52 A similar movement has been detected with PELDOR in a related hyperpolarization-activated and cyclic nucleotide-gated channel (HCN2) 53 and the bacterial CNG channel SthK. 54 We will show that MHQ/PELDOR can resolve conformational changes of the MloK1 CNBD on the angstrom and lowmicrosecond time scale.…”

“…This picture agrees with recent atomistic simulations, which revealed "prebinding" of the ligand to different surface sites, followed by induced-fit conformational motions of the binding pocket and entropic barriers to ligand binding as the ratelimiting steps. 52 We note that this concept neither rules out conformational motions during the first ligand binding steps that, however, are below the PELDOR resolution, nor claims that, for the apo-ligand complex, the ligand is already positioned at its final binding site. It does imply, though, that the second step is independent of concentration.…”

“…Here we study the ligand-induced conformational dynamics of the bacterial cyclic nucleotide-gated (CNG) K + channel from Mesorhizobium loti (MloK1), which opens by binding of cyclic adenosine monophosphate (cAMP) to a cyclic nucleotide-binding domain (CNBD). , Upon binding, the CNBD undergoes a conformational change, including a movement of the C-terminal C α -helix (Figure ). This movement has been inferred from X-ray and NMR , structures of the CNBD in its apo and holo states and has also been predicted by atomistic simulations . A similar movement has been detected with PELDOR in a related hyperpolarization-activated and cyclic nucleotide-gated channel (HCN2) and the bacterial CNG channel SthK …”