Human carbonic anhydrases (HCAs) are responsible for pH control and sensing in our body and constitute key components in the central pH paradigm connected to cancer therapeutics. Of the 12 catalytically active HCA isoforms expressed in humans, the isozymes HCA IX and HCA XII have been implicated in cancer. The major objective of our study is to use computer simulation to obtain a molecular-level understanding of HCA IX because of its high expression in solid tumours and its role in controlling the pH of the tumour microenvironment including tumour cell survival and proliferation.

HCA IX is a multi-domain, dimeric trans-membrane enzyme for which only the catalytic domain (labelled as HCA IX-c) has been crystallized. Starting from the crystal structure, we have carried out constant pH MD (CpHMD) simulation of both monomeric and dimeric HCA IX-c in water at pH = 5.0, 6.0, 7.0 and 8.0. At each pH, a 1 μs long CpHMD trajectory is combined with umbrella sampling, transition path sampling and Markov state models to probe the pH-dependent stability and functional dynamics of HCA IX. The results of our simulations highlight how pH modulates catalytically important factors such as fluctuations of a proton shuttling histidine residue and reorganization of active site water molecules to form a proton path for the rate-determining intramolecular proton transfer reaction. The histidine residue was found to fluctuate between two conformations at all pH values, but their relative stability and the underlying free energy surfaces depend strongly on the solvent pH. The kinetic rate constants of inter-conversion between different free energy basins are found to span 10⁷-10⁹ s^-1. We also investigate how the alteration of proton shuttle and hydrogen-bonded connectivity depend on whether the catalytic domain is mono- or dimeric thereby highlighting the interplay of allostery and pH in determining the structure and function of HCA IX.

(a) Hydration of the active site of monomeric HCA IX-c with 50% occupation probability of water molecules around Zn(II) ion. (b) Unweighted free energy landscape obtained from tICA for monomeric (top) and dimeric (bottom) HCA IX-c.

Divya Rai

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