Molecular dynamics study of human carbonic anhydrase II in complex with Zn2+ and acetazolamide on the basis of all-atom force field simulations
Graphical abstract
Introduction
Carbonic anhydrases (CAs), [1] a ubiquitous group of zinc-bound proteins (metalloenzymes) [2], [3], have long been known to catalyze the hydration of carbon dioxide at an extremely high rate [4]. This colorless metalloprotein has one zinc ion per molecule [5], but a substitution of zinc with cobalt, in bovine carbonic anhydrase, gives a colored enzyme with 45% of the native enzyme activity [6]. Human carbonic anhydrase II (hCAII), in particular, is a cytosolic and monomeric protein with a molecular weight of 30 kDa [7]. Its tertiary structure (illustrated in Fig. 1) is represented by a 15 Å deep conical binding pocket [8] formed largely with 10-stranded beta-sheets, [9] which is lined, on one side, with hydrophobic residues and, on the other side, with hydrophilic residues. At the bottom of the pocket, a zinc ion is tetrahedrally coordinated with three histidine residues (HIS 94, 96 and 119) and a bound water/hydroxyl (H2O/OH–) or the NH group of an inhibitor, acetazolamide (AZM), lodged in the pocket of the enzyme's active site [10].
Human carbonic anhydrase II plays very fundamental roles in human physiology/pathology. It is essential in keeping the adequate balance between carbon dioxide and bicarbonate and thus controlling the pH level in cells. Mutations of hCAII have been found to cause carbonic anhydrase deficiency syndrome [13], [14], [15] leading to diseases such as osteopetrosis, renal tubular acidosis [13], [16], and cerebral calcification [17], [18]. On the other hand, overexpression of hCAII was found to favor glaucoma [19]. An inhibitor of hCAII, N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide (namely, acetazolamide), was found to bind hCAII with a dissociation constant of 6.8 nM [20], [21] and an inhibition constant in the range of 3.3–12 nM [22], [23], [24], [25]. Near the physiological pH, AZM has multiple protonation states (shown in Fig. 2). Two pKa values (7.19 and 8.65) were experimentally determined [26], [27], [28]. These four and other protonation states can coexist as determined by several factors [28]. It should be noted that the protonation state of AZM in complex with hCAII was revealed in a very interesting recent experiment employing the technique of neutron diffraction [26]. State (AZM-1a) of Fig. 2 was the observed state, which is consistent with the fact that Zn2+ very effectively lowers the pKa of even H2O. Zn2+ can readily force the deprotonation of the sulfamoyl group when a neutral AZM falls into the binding pocket of hCAII (Fig. 1).
Although numerous experimental studies are available on hCAII and its inhibitors, systematic molecular dynamic (MD) simulations have not yet been done to gain the atomistic, dynamics insights and quantitative understanding of the interactions between hCAII and the available inhibitors. One of the difficulties for the theoretical/computational studies is how to accurately represent the key role played by the metal ion Zn2+. Now we have the experimentally determined values of the AZM-hCAII binding affinity, the structure of the AZM-hCAII complex resolved to atomistic resolutions, and even the protonation state of the metalloenzyme complex. It is time for us to obtain a quantitative computational/theoretical understanding of this fundamentally important metalloprotein-inhibitor complex in a systematic and self-consistent investigation. It is needless to note that a good understanding of these interactions will be helpful in the pursuit of new or improved inhibitors/modulators of hCAII and other carbonic anhydrases.
In this article, we present a detailed, quantitative characterization of the AZM-hCAII-Zn2+ binding problems on the basis of all-atom MD simulations. First, we address one of the currently debated issues by testing the non-bonded model of Zn2+, in which the metal ion is simply represented by two van der Waals parameters. With such a simple model, we compute the free energy of hydration of Zn2+. We further compute the binding characteristics of Zn2+ to the hCAII protein in the uninhibited and inhibited states. In all cases, our computed values agree with the experimental measurements when we use the recently refined Zn2+ parameters of Li et al. [29] along with the standard CHARMM parameters other than those for the divalent metal ion. Then, we compute the free energies of binding the charged form (State AZM-1a, Fig. 2) and the neutral form (State AZM0, Fig. 2) of AZM to hCAII. In this comparative study, we provide detailed, quantitative insights about the binding interactions between AZM-1a, Zn2+, and hCAII, all in agreement with the experimental data within the known margin of error.
Section snippets
Simulation parameters
In all the equilibrium MD and nonequilibrium steered molecular dynamics (SMD) runs, we use the CHARMM36 force field [30], [31] for all the intra- and inter-molecular interactions except for Zn2+. For Zn2+, we use two sets of van der Waals (vdW) parameters (shown in Table 1) in the computation of its free energy of hydration. On the basis of a better agreement between our computed and the experimentally measured values of the hydration free energy, we choose the recently refined parameter set of
H2O-Zn2+ interactions
Foremost among all ion interactions in a biological system are the interactions with water molecules. For the purpose of choosing an optimum set of parameters, we computed the free energy of zinc hydration using both sets of parameters in Table 1. The PMF curves along the Zn2+ hydration path are shown in Fig. 4(A). Note that, from z = ∞ to zA = 10 Å, the reversible work [Eq. (1)] is equal to − 32.7 kcal/mol. Combining this analytical approximation with the numerical values in Fig. 4(A), the computed
Conclusions
On the basis of the all-atom CHARMM force field parameters including the recently refined zinc parameters, our extensive simulations lead us to these conclusions: The key biophysical/biochemical interactions of the divalent zinc ion with the aqueous environmental factors, with the protein, and with the hCAII inhibitor, AZM, can all be well approximated with a simple ion model using the refined vdW parameters of Li et al. [29] The existence of the divalent zinc ion at the active site causes the
Acknowledgements
The authors are thankful to Dr. Robert Renthal for his biochemical insights. They acknowledge the NIH grants GM084834 and GM060655. They also acknowledge the Texas Advanced Computing Center at the University of Texas at Austin for the supercomputing time grants.
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