The influences of protonation state of histidine on aromatic/arginine region of aquaporin-1 protein

Aquaporin-1 (AQP1) is widely distributed in the epithelial tissue for water absorption and secretion. The histidine (His182) residue in the aromatic/arginine (ar/R) constriction region plays an important role in transporting water through the membrane. In this study, we have performed a total of 46 ns equilibrium molecular dynamics (MD) simulations, and obtained the influence of His182 in two protonation states (Hsd is the proton at N_δ and Hse is the proton at N_?) on the ar/R region. Water permeation rate shows that it is easier for water molecules to permeate the ar/R region of the AQP1 with residue in the Hsd state than in the Hse state. The minimum radii of the pore in the ar/R region were calculated during the last 10 ns MD simulation. We have analysed the correlation among the state of the pore (open or close), the minimum radius of the ar/R region and the dihedral angles < C_β-C_γ-C_δ-N_? of Arg197. The results show that the minimum radius can be used to mark the state of the pore.     

Impact of ligand and protein desolvation on ligand binding to the S1 pocket of thrombin

In the present study, we investigate the impact of a tightly bound water molecule on ligand binding in the S1 pocket of thrombin. The S1 pocket contains a deeply buried deprotonated aspartate residue (Asp189) that is, due to its charged state, well hydrated in the uncomplexed state. We systematically studied the importance of this water molecule by evaluating a series of ligands that contains pyridine-type P1 side chains that could potentially alter the binding properties of this water molecule. All of the pyridine derivatives retain the original hydration state albeit sometimes with a slight perturbance. In order to prevent a direct H-bond formation with Asp189, and to create a permanent positive charge on the P1 side chain that is positioned adjacent to the Asp189 carboxylate anion, we methylated the pyridine nitrogen. This methylation resulted in displacement of water but was accompanied by a loss in binding affinity. Quantum chemical calculations of the ligand solvation free energy showed that the positively charged methylpyridinium derivatives suffer a large penalty of desolvation upon binding. Consequently, they have a substantially less favorable enthalpy of binding. In addition to the ligand desolvation penalty, the hydration shell around Asp189 has to be overcome, which is achieved in nearly all pyridinium derivatives. Only for the ortho derivative is a partial population of a water next to Asp189 found. Possibly, the gain of electrostatic interactions between the charged P1 side chain and Asp189 helps to compensate for the desolvation penalty. In all uncharged pyridine derivatives, the solvation shell remains next to Asp189, partly mediating interactions between ligand and protein. In the case of the para-pyridine derivative, a strongly disordered cluster of water sites is observed between ligand and Asp189.     

Enhancement of proton conductance by mutations of the selectivity filter of aquaporin-1

Prevention of cation permeation in wild-type aquaporin-1 (AQP1) is believed to be associated with the Asn-Pro-Ala (NPA) region and the aromatic/arginine selectivity filter (SF) domain. Previous work has suggested that the NPA region helps to impede proton permeation due to the protein backbone collective macrodipoles that create an environment favoring a directionally discontinuous channel hydrogen-bonded water chain and a large electrostatic barrier. The SF domain contributes to the proton permeation barrier by a spatial restriction mechanism and direct electrostatic interactions. To further explore these various effects, the free-energy barriers and the maximum cation conductance for the permeation of various cations through the AQP1-R195V and AQP1-R195S mutants are predicted computationally. The cations studied included the hydrated excess proton that utilizes the Grotthuss shuttling mechanism, a model “classical” charge localized hydronium cation that exhibits no Grotthuss shuttling, and a sodium cation. The hydrated excess proton was simulated using a specialized multi-state molecular dynamics method including a proper physical treatment of the proton shuttling and charge defect delocalization. Both AQP1 mutants exhibit a surprising cooperative effect leading to a reduction in the free-energy barrier for proton permeation around the NPA region due to altered water configurations in the SF region, with AQP1-R195S having a higher conductance than AQP1-R195V. The theoretical predictions are experimentally confirmed in wild-type AQP1 and the mutants expressed in Xenopus oocytes. The combined results suggest that the SF domain is a specialized structure that has evolved to impede proton permeation in aquaporins.     

Pore-forming transmembrane domains control ion selectivity and selectivity filter conformation in the KirBac1.1 potassium channel

Potassium (K⁺) channels are membrane proteins with the remarkable ability to very selectively conduct K⁺ ions across the membrane. High-resolution structures have revealed that dehydrated K⁺ ions permeate through the narrowest region of the pore, formed by the backbone carbonyls of the signature selectivity filter (SF) sequence TxGYG. However, the existence of nonselective channels with similar SF sequences, as well as effects of mutations in other regions on selectivity, suggest that the SF is not the sole determinant of selectivity. We changed the selectivity of the KirBac1.1 channel by introducing mutations at residue I131 in transmembrane helix 2 (TM2). These mutations increase Na⁺ flux in the absence of K⁺ and introduce significant proton conductance. Consistent with K⁺ channel crystal structures, single-molecule FRET experiments show that the SF is conformationally constrained and stable in high-K⁺ conditions but undergoes transitions to dilated low-FRET states in high-Na⁺/low-K⁺ conditions. Relative to wild-type channels, I131M mutants exhibit marked shifts in the K⁺ and Na⁺ dependence of SF dynamics to higher K⁺ and lower Na⁺ concentrations. These results illuminate the role of I131, and potentially other structural elements outside the SF, in controlling ion selectivity, by suggesting that the physical interaction of these elements with the SF contributes to the relative stability of the constrained K⁺-induced SF configuration versus nonselective dilated conformations.     

Dynamic water networks in cytochrome cbb(3) oxidase

Heme-copper oxidases (HCOs) are terminal electron acceptors in aerobic respiration. They catalyze the reduction of molecular oxygen to water with concurrent pumping of protons across the mitochondrial and bacterial membranes. Protons required for oxygen reduction chemistry and pumping are transferred through proton uptake channels. Recently, the crystal structure of the first C-type member of the HCO superfamily was resolved [Buschmann et al. Science 329 (2010) 327-330], but crystallographic water molecules could not be identified. Here we have used molecular dynamics (MD) simulations, continuum electrostatic approaches, and quantum chemical cluster calculations to identify proton transfer pathways in cytochrome cbb(3). In MD simulations we observe formation of stable water chains that connect the highly conserved Glu323 residue on the proximal side of heme b(3) both with the N- and the P-sides of the membrane. We propose that such pathways could be utilized for redox-coupled proton pumping in the C-type oxidases. Electrostatics and quantum chemical calculations suggest an increased proton affinity of Glu323 upon reduction of high-spin heme b(3). Protonation of Glu323 provides a mechanism to tune the redox potential of heme b(3) with possible implications for proton pumping.     

Water and ion permeation in bAQP1 and GlpF channels: a kinetic Monte Carlo study

The kinetic Monte Carlo reaction-path-following technique is applied to determine the lowest-energy water pathway and the coordinating amino acids in bAQP1 and GlpF channels, both treated as rigid. In bAQP1, water molecules pass through the pore between the asparagine-proline-alanine (NPA) and selectivity filter (SF) sites one at a time. The water chain is interrupted at the SF where one water forms three stable hydrogen bonds with protein atoms. In this SF, water's conformation depends on the protonation locus of H182. In GlpF, two water molecules bond simultaneously to the NPA asparagines and pass through the SF in zigzag fashion. No water single-file forms in rigid GlpF. To accommodate a single file of waters requires narrowing the GlpF pore. Our results reveal that in both proteins a proposed bipolar water arrangement is thermally disrupted in the NPA region, especially in the cytoplasmic part of the pore. The equilibrium hydrogen-bonded chain is occasionally interrupted in the hydrophobic zones adjacent to the NPA motifs. The permeation of alkali cations through bAQP1 and GlpF is barred due to a large free-energy barrier in the NPA region as well as a large energy barrier blocking entry from the cytoplasm. Permeation of halides is prevented due to two large energy barriers in the cytoplasmic and periplasmic pores as well as a large free-energy barrier barring entry from the periplasm. Our results, based on modeling charge permeation, support an electrostatic rather than orientational basis for proton exclusion. Binding within the aquaporin pore cannot compensate sufficiently for dehydration of the protonic charge; there is also an electrostatic barrier in the NPA region blocking proton transport. The highly ordered single file of waters, which is drastically interrupted at the SF of bAQP1, may also contribute to proton block.     

The ionization state and the conformation of Glu-71 in the KcsA K(+) channel.

The side chain of Glu-71 of the KcsA K(+) channel, an important residue in the vicinity of the selectivity filter, was not resolved in the crystallographic structure of Doyle et al. (Doyle, D. A., J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, and R. MacKinnon. 1998. Science. 280:69-77). Its atomic coordinates are undetermined and its ionization state is unknown. For meaningful theoretical and computational studies of the KcsA K(+) channel, it is essential to address questions about the conformation and the ionization state of this residue in detail. In previous MD simulations in which the side chain of Glu-71 is protonated and forming a strong hydrogen bond with Asp-80 it was observed that the channel did not deviate significantly from the crystallographic structure (Bernèche, S., and B. Roux. 2000. Biophys. J. 78:2900-2917). In contrast, we show here that the structure of the selectivity filter of the KcsA channel is significantly disrupted when these side chains are fully ionized on each of the four monomers. To further resolve questions about the ionization state of Glu-71 we calculated the pK(a) value of this residue using molecular dynamics free energy simulations (MD/FES) with a fully flexible system including explicit solvent and membrane and finite-difference Poisson-Boltzmann (PB) continuum electrostatics. It is found that the pK(a) of Glu-71 is shifted by approximately +10 pK(a) units. These results strongly suggest that Glu-71 is protonated under normal conditions.     

On the dependence of molecular conformation on the type of solvent environment: a molecular dynamics study of cyclosporin A

The dependence of the conformation of cyclosporin A (CPA), a cyclic undecapeptide with potent immunosuppressive activity, on the type of solvent environment is examined using the computer simulation method of molecular dynamics (MD). Conformational and dynamic properties of CPA in aqueous solution are obtained from MD simulations of a CPA molecule dissolved in a box with water molecules. Corresponding properties of CPA in apolar solution are obtained from MD simulations of CPA in a box with carbontetrachloride. The results of these simulations in H2O and in CCl4 are compared to each other and to those of previous simulations of crystalline CPA and of an isolated CPA molecule. The conformation of the backbone of the cyclic polypeptide is basically independent of the type of solvent. In aqueous solution the beta-pleated sheet is slightly weaker and the gamma-turn is a bit less pronounced than in apolar solution. Side chains may adopt different conformations in different solvents. In apolar solution the hydrophobic side chain of the MeBmt residue is in an extended conformation with its hydroxyl group hydrogen bonded to the backbone carbonyl group. In aqueous solution this hydrophobic side chain folds over the core of the molecule and the mentioned hydrogen bond is broken in favor of hydrogen bonding to water molecules. The conformation obtained from the MD simulation in CCl4 nicely agrees with experimental atom-atom distance data as obtained from nmr experiments in chloroform. In aqueous solution the relaxation of atomic motion tends to be slower than in apolar solution.     

Aquaporin gating

An acceleration in the rate at which new aquaporin structures are determined means that structural models are now available for mammalian AQP0, AQP1, AQP2 and AQP4, bacterial GlpF, AqpM and AQPZ, and the plant SoPIP2;1. With an apparent consensus emerging concerning the mechanism of selective water transport and proton extrusion, emphasis has shifted towards the issues of substrate selectivity and the mechanisms of aquaporin regulation. In particular, recently determined structures of plant SoPIP2;1, sheep and bovine AQP0, and Escherichia coli AQPZ provide new insights into the underlying structural mechanisms by which water transport rates are regulated in diverse organisms. From these results, two distinct pictures of 'capping' and 'pinching' have emerged to describe aquaporin gating.     

Functional plasticity in the substrate binding site of beta-secretase

The aspartic protease beta-secretase (BACE) cleaves the amyloid precursor protein into a 42 residue beta-peptide, which is the principal biochemical marker of Alzheimer's disease. Multiple explicit-water molecular dynamics simulations of the apo and inhibitor bound structures of BACE indicate that both open- and closed-flap conformations are accessible at room temperature and should be taken into account for inhibitor design. Correlated motion is observed within each of the two lobes of BACE, as well as for the interfacial region. A self-inhibited conformation with the side chain of Tyr71 occupying the S(1) pocket is present in some of the unbound simulations. The reversible loss of the side chain hydrogen bond between the catalytic Asp32 and Ser35, due to the concomitant reorientation of the Ser35 hydroxyl group and a water molecule conserved in pepsin-like enzymes, provides further evidence for the suggestion that Ser35 assists in proton acceptance and release by Asp32 during catalysis.     

Redesign of the proton-pumping machinery of cytochrome c oxidase: proton pumping does not require Glu(I-286)

One of the putative proton-transfer pathways leading from solution toward the binuclear center in many cytochrome c oxidases is the D-pathway, so-called because it starts with a highly conserved aspartate [D(I-132)] residue. Another highly conserved amino acid residue in this pathway, glutamate(I-286), has been indicated to play a central role in the proton-pumping machinery of mitochondrial-type enzymes, a role that requires a movement of the side chain between two distinct positions. In the present work we have relocated the glutamate to the opposite side of the proton-transfer pathway by constructing the double mutant EA(I-286)/IE(I-112). This places the side chain in about the same position in space as in the original enzyme, but does not allow for the same type of movement. The results show that the introduction of the second-site mutation, IE(I-112), in the EA(I-286) mutant enzyme results in an increase of the enzyme activity by a factor of >10. In addition, the double mutant enzyme pumps approximately 0.4 proton per electron. This observation restricts the number of possible mechanisms for the operation of the redox-driven proton pump. The proton-pumping machinery evidently does require the presence of a protonatable/polar residue at a specific location in space, presumably to stabilize an intact water chain. However, this residue does not necessarily have to be at a strictly conserved location in the amino acid sequence. In addition, the results indicate that E(I-286) is not the "proton gate" of cytochrome c oxidase controlling the flow of pumped protons from one to the other side of the membrane.     

Atomic resolution structures of bacteriorhodopsin photocycle intermediates: the role of discrete water molecules in the function of this light-driven ion pump

High-resolution X-ray crystallographic studies of bacteriorhodopsin have tremendously advanced our understanding of this light-driven ion pump during the last 2 years, and emphasized the crucial role of discrete internal water molecules in the pump cycle. In the extracellular region an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base via water 402 and the initial proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a pi-bulge that causes a non-proline kink. The bulge is stabilized by hydrogen bonding of the main chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located in the otherwise very hydrophobic region between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The M intermediate trapped in the D96N mutant corresponds to a late M state in the transport cycle, after protonation of Asp85 and release of a proton to the extracellular membrane surface, but before reprotonation of the deprotonated retinal Schiff base. The M intermediate from the E204Q mutant corresponds to an earlier M, as in this mutant the Schiff base deprotonates without proton release. The structures of these two M states reveal progressive displacements of the retinal, main chain and side chains induced by photoisomerization of the retinal to 13-cis,15-anti, and an extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pK(a)s of the Schiff base, Asp85, the proton release group and Asp96. The structure for the M state from E204Q suggests, moreover, that relaxation of the steric conflicts of the distorted 13-cis,15-anti retinal plays a critical role in the reprotonation of the Schiff base by Asp96. Two additional waters now connect Asp96 to the carbonyl of residue 216, in what appears to be the beginning of a hydrogen-bonded chain that would later extend to the retinal Schiff base. Based on the ground state and M intermediate structures, models of the molecular events in the early part of the photocycle are presented, including a novel model which proposes that bacteriorhodopsin pumps hydroxide (OH(-)) ions from the extracellular to the cytoplasmic side.     

Comparative simulations of aquaporin family: AQP1, AQPZ, AQP0 and GlpF

Molecular dynamics simulations were performed for four members of the aquaporin family (AQP1, AQPZ, AQP0, and GlpF) in the explicit membrane environment. The single-channel water permeability, pf, was evaluated to be GlpF approximately AQPZ > AQP1 > AQP0, while their relative pore sizes were GlpF > AQP1 > AQPZ > AQP0. This relation between pf and pore size indicates that water permeability was determined not only by the channel radius, but also another competing factor. Analysis of water dynamics revealed that this factor was the single-file nature of water transport.     

Molecular dynamics of the KcsA K(+) channel in a bilayer membrane

Molecular dynamics (MD) simulations of an atomic model of the KcsA K(+) channel embedded in an explicit dipalmitoylphosphatidylcholine (DPPC) phospholipid bilayer solvated by a 150 mM KCl aqueous salt solution are performed and analyzed. The model includes the KcsA K(+) channel, based on the recent crystallographic structure of, Science. 280:69-77), 112 DPPC, K(+) and Cl(-) ions, as well as over 6500 water molecules for a total of more than 40,000 atoms. Three K(+) ions are explicitly included in the pore. Two are positioned in the selectivity filter on the extracellular side and one in the large water-filled cavity. Different starting configurations of the ions and water molecules in the selectivity filter are considered, and MD trajectories are generated for more than 4 ns. The conformation of KcsA is very stable in all of the trajectories, with a global backbone root mean square (RMS) deviation of less than 1.9 A with respect to the crystallographic structure. The RMS atomic fluctuations of the residues surrounding the selectivity filter on the extracellular side of the channel are significantly lower than those on the intracellular side. The motion of the residues with aromatic side chains surrounding the selectivity filter (Trp(67), Trp(68), Tyr(78), and Tyr(82)) is anisotropic with the smallest RMS fluctuations in the direction parallel to the membrane plane. A concerted dynamic transition of the three K(+) ions in the pore is observed, during which the K(+) ion located initially in the cavity moves into the narrow part of the selectivity filter, while the other two K(+) ions move toward the extracellular side. A single water molecule is stabilized between each pair of ions during the transition, suggesting that each K(+) cation translocating through the narrow pore is accompanied by exactly one water molecule, in accord with streaming potential measurements (, Biophys. J. 55:367-371). The displacement of the ions is coupled with the structural fluctuations of Val(76) and Gly(77), in the selectivity filter, as well as the side chains of Glu(71), Asp(80), and Arg(89), near the extracellular side. Thus the mechanical response of the channel structure at distances as large as 10-20 A from the ions in the selectivity filter appears to play an important role in the concerted transition.     

Dynamic water networks in cytochrome cbb3 oxidase

Heme-copper oxidases (HCOs) are terminal electron acceptors in aerobic respiration. They catalyze the reduction of molecular oxygen to water with concurrent pumping of protons across the mitochondrial and bacterial membranes. Protons required for oxygen reduction chemistry and pumping are transferred through proton uptake channels. Recently, the crystal structure of the first C-type member of the HCO superfamily was resolved [Buschmann et al. Science 329 (2010) 327–330], but crystallographic water molecules could not be identified. Here we have used molecular dynamics (MD) simulations, continuum electrostatic approaches, and quantum chemical cluster calculations to identify proton transfer pathways in cytochrome cbb₃. In MD simulations we observe formation of stable water chains that connect the highly conserved Glu323 residue on the proximal side of heme b₃ both with the N- and the P-sides of the membrane. We propose that such pathways could be utilized for redox-coupled proton pumping in the C-type oxidases. Electrostatics and quantum chemical calculations suggest an increased proton affinity of Glu323 upon reduction of high-spin heme b₃. Protonation of Glu323 provides a mechanism to tune the redox potential of heme b₃ with possible implications for proton pumping.     

Permeability properties of ENaC selectivity filter mutants.

The epithelial Na(+) channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na(+) absorption. The amiloride-sensitive ENaC is highly selective for Na(+) and Li(+) ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alphaS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K(+) ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alphaS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha589, indicating that alphaS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na(+) and Li(+). These findings demonstrate the critical role of the pore size at the alphaS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the alphaS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alphaS589 side chain is oriented toward the subunit-subunit interface and that substitution of alphaS589 by larger residues increases the pore diameter by adding extra volume at the subunit-subunit interface.     

Allosteric coupling between transmembrane segment 4 and the selectivity filter of TALK1 potassium channels regulates their gating by extracellular pH

Opening of two-pore domain K⁺ channels (K2Ps) is regulated by various external cues, such as pH, membrane tension, or temperature, which allosterically modulate the selectivity filter (SF) gate. However, how these cues cause conformational changes in the SF of some K2P channels remains unclear. Herein, we investigate the mechanisms by which extracellular pH affects gating in an alkaline-activated K2P channel, TALK1, using electrophysiology and molecular dynamics (MD) simulations. We show that R233, located at the N-terminal end of transmembrane segment 4, is the primary pH_o sensor. This residue distally regulates the orientation of the carbonyl group at the S1 potassium-binding site through an interacting network composed of residues on transmembrane segment 4, the pore helix domain 1, and the SF. Moreover, in the presence of divalent cations, we found the acidic pH-activated R233E mutant recapitulates the network interactions of protonated R233. Intriguingly, our data further suggested stochastic coupling between R233 and the SF gate, which can be described by an allosteric gating model. We propose that this allosteric model could predict the hybrid pH sensitivity in heterodimeric channels with alkaline-activated and acidic-activated K2P subunits.     

The mechanism of proton translocation in respiratory complex I from molecular dynamics

Abstract

Respiratory complex I, the biggest enzyme of respiratory chain, plays a key role in energy production by the mitochondrial respiratory chain and has been implicated in many human neurodegenerative diseases. Recently, the crystal structure of respiratory complex I is reported. We perform 50?ns molecular dynamics simulations on the membrane domain of respiratory complex I under two hypothetical states (oxidized state and reduced state). We find that the density of water molecules in the trans-membrane domain under reduced state is bigger than that under oxidized state. The connecting elements (helix HL and β-hairpins-helix element) fluctuate stronger under reduced state than that under oxidized state, causing more internal water molecules and facilitating the proton conduction. The conformational changes of helix HL and the crucial charged residue Glu in TM5 play key roles in the mechanism of proton translocation. Our results illustrate the dynamic behavior and the potential mechanism of respiratory complex I, which provides the structural basis for drug design of respiratory complex I.     

Crystal structure of AqpZ tetramer reveals two distinct Arg-189 conformations associated with water permeation through the narrowest constriction of the water-conducting channel

AqpZ is a homotetramer of four water-conducting channels that facilitate rapid water movements across the plasma membrane of Escherichia coli. Here we report a 3.2 angstroms crystal structure of the tetrameric AqpZ (tAqpZ). All channel-lining residues in the four monomeric channels are found orientated in nearly identical positions with one marked exception at the narrowest channel constriction, where the side chain of a highly conserved Arg-189 adopts two distinct conformational orientations. In one of the four monomers, the guanidino group of Arg-189 points toward the periplasmic vestibule, opening up the constriction to accommodate the binding of a water molecule through a tridentate H-bond. In the other three monomers, the Arg-189 guanidino group bends over to form an H-bond with carbonyl oxygen of the Thr-183, thus occluding the channel. Therefore, the tAqpZ structure reveals two distinct Arg-189 confirmations associated with water permeation through the channel constrictions. Alternation between the two Arg-189 conformations disrupts continuous flow of water, thus regulating the open probability of the water pore. Further, the difference in Arg-189 displacements is correlated with a strong electron density found between the first transmembrane helices of two open channels, suggesting that the observed Arg-189 conformations are stabilized by asymmetrical subunit interactions in tAqpZ.     

Atomic crystal and molecular dynamics simulation structures of human carbonic anhydrase II: insights into the proton transfer mechanism

Human carbonic anhydrase II (HCA II) is a zinc-metalloenzyme that catalyzes the reversible interconversion of CO2 and HCO3-. The rate-limiting step of this catalysis is the transfer of a proton between the Zn-bound solvent molecule and residue His64. In order to fully characterize the active site structural features implicated in the proton transfer mechanism, the refined X-ray crystal structure of uncomplexed wild type HCA II to 1.05 A resolution with an Rcryst value of 12.0% and an Rfree value of 15.1% has been elucidated. This structure provides strong clues as to the pathway of the intramolecular proton transfer between the Zn-bound solvent and His64. The structure emphasizes the role of the solvent network, the unique positioning of solvent molecule W2, and the significance of the dual conformation of His64 in the active site. The structure is compared with molecular dynamics (MD) simulation calculations of the Zn-bound hydroxyl/His64+ (charged) and the Zn-bound water/His64 (uncharged) HCA II states. A comparison of the crystallographic anisotropic atomic thermal parameters and MD simulation root-mean-square fluctuation values show excellent agreement in the atomic motion observed between the two methods. It is also interesting that the observed active site solvent positions in the crystal structure are also the most probable positions of the solvent during the MD simulations. On the basis of the comparative study of the MD simulation results, the HCA II crystal structure observed is most likely in the Zn-bound water/His64 state. This conclusion is based on the following observations: His64 is mainly (80%) orientated in an inward conformation; electron density omit maps infer that His64 is not charged in an either inward or outward conformation; and the Zn-bound solvent is most likely a water molecule.