Structure and Dynamics of the Isozymes II and IX of Human Carbonic Anhydrase

Abstract

Human carbonic anhydrases (HCAs) are responsible for the pH control and sensing in our body and constitute key components in the central pH paradigm connected to cancer therapeutics. However, little or no molecular level studies are available on the pH-dependent stability and functional dynamics of the known isozymes of HCA. The main objective of this Article is to report the first bench-marking study on the structure and dynamics of the two most efficient isozymes, HCA II and IX, at neutral pH using classical molecular dynamics (MD) and constant pH MD (CpHMD) simulations combined with umbrella sampling, transition path sampling, and Markov state models. Starting from the known crystal structures of HCA II and the monomeric catalytic domain of HCA IX (labeled as HCA IX-c), we have generated classical MD and CpHMD trajectories (of length 1 μs each). In all cases, the overall stability, RMSD, and secondary structure segments of the two isozymes are found to be quite similar. Functionally important dynamics of these two enzymes have been probed in terms of active site hydration, coordination of the Zn(II) ion to a transient excess water, and the formation of putative proton transfer paths. The most important difference between the two isozymes is observed for the side-chain fluctuations of His-64 that is expected to shuttle an excess proton out of the active site as a part of the rate-determining intramolecular proton transfer reaction. The relative stability of the stable inward and outward conformations of the His-64 side-chain and the underlying free energy surfaces are found to depend strongly on the isozyme. In each case, a lower free energy barrier is detected between predominantly inward conformations from predominantly outward ones when simulated under constant pH conditions. The kinetic rate constants of interconversion between different free energy basins are found to span 10 7 –10 8 s –1 with faster conformational transitions predicted at constant pH condition. The estimated rate constants and free energies are expected to validate if the fluctuation of the His-64 side-chain in HCA IX may have a significance similar to that known in the multistep catalytic cycle of HCA II.

pH-Dependent Structure and Dynamics of the Catalytic Domains of Human Carbonic Anhydrase II and IX

“…But the reorganization dynamics at the active site, as presented in Figure c, was significantly affected by the use of a constant-pH condition. For the conformational fluctuation of His-64 side chain in both HCA II and IX-c, the slowest time scale was estimated to be 1 order of magnitude slower than that observed under charge-neutralized condition of conventional MD …”

“…Several short dynamical trajectories should ideally be employed to construct the time-lagged MSM and obtain statistically reliable estimates of free energy and kinetics associated with the transitions of interest. Our earlier work at neutral pH indicated that the rate constants of transition between in and out conformations of His-64 are in the range of 10 8 –10 7 s –1 . Accordingly, we employed a single long CpHMD trajectory of length 1 μs at each pH for each isozyme in the present work.…”

“…The computational strategy to mimic this effect in silico involves allowing dynamic fluctuations between all charged states of titrable amino acid residues (Asp, Glu, His, Cys, Lys, Tyr) and adding coion(s) to maintain the overall electroneutrality of the simulated system. , We then perform constant-pH MD (CpHMD) simulation for HCA IX-c and II in explicit water at different values of pH. In an earlier work, we have reported an extensive bench-marking study on the use of CpHMD simulation on these two isozymes at pH = 7 . A major success in this study was to generate stable, solvated structures of both isozymes at constant pH.…”

“…In an earlier work, we have reported an extensive bench-marking study on the use of CpHMD simulation on these two isozymes at pH = 7. 13 A major success in this study was to generate stable, solvated structures of both isozymes at constant pH. In particular, an initial structure of HCA IX-c (PDB: 6FE2 15 ), crystallized at pH 4.5, could be utilized to simulate its structure and dynamics at pH 7.…”

“…Both are thermally stable with melting temperatures, T M ∼56–59 °C at pH = 7 . However, they have only about 31% sequence identity and differ most notably from each other in their respective pH-dependent expression and catalytic activity. ,, In the present paper, we report, for the first time, a molecular perspective of how their structure and functional dynamics respond to changes in pH from 5 to 8 from detailed simulation studies on HCA II and the catalytic domain of HCA IX (HCA IX-c) in water. Our major objective is to correlate the molecular level analysis with known experimental studies on these two isozymes.…”