Molecular dynamics study of the structural role of metal atoms in the urease active site

Abstract

Introduction.Urease is a representative of a small group of enzymes that can bind different alternative metals to execute the same catalytic function. The experimental X-ray studies conclude that a urease activity critically depends on the precise positions of amino acid ligands at a metallocenter, the bound solvent molecules and the type of metal, and very subtle changes of metallocenter structure can essentially influence the urease activity. Are these conclusions valid in the case of the urease structures in the solution? By molecular dynamics simulations, we studied these aspects for urease derivatives with alternative metals in solution under physiological pH and temperatures. Methods.Moleculardynamics (MD)simulationswerecarriedoutforthe following systems: Ni-containingSporosarcina pasteurii ureasebothnative (PDBcode 2ubp) andincomplexwithcompetitiveinhibitoracetohydroxamicacid, AHA (PDBcode 4ubp); Klebsiellaaerogenes urease both Ni- andMn-containing(PDBcodes 1fwjand 1ef2, respectively); Fe-containingHelicobactermustelaeurease (PDB code 3qga) (table 1). As well, there was studied an apoenzyme of Ni-containing urease: the structure of the complex of competitive inhibitor AHA with Sporosarcina pasteurii urease (4ubp) from which there were removed Ni atoms. Thesystemsstudied (α subunitsoritscomplexwithcompetitiveinhibitor) wereplacedina cubic periodic cell filled with TIP3P water molecules. The simulation cell was 1 nm larger than the molecular system studied along all three axes. Na+ and Cl¯ counterions were added to neutralize the system and to reach ion mass fraction 0.9% NaCl. Before simulations the systems were energy-minimized. After a short steepest descent minimization, the the procedure continued by simulated annealing minimization.  AMBER14ipq force field was used. To treat long-range electrostatic interactions the Particle Mesh Ewald algorithm was used. The equations of the movement were integrated by 2.5 fs step. Tospeed upthe calculations thenon-boundedvanderWaalsandelectrostatic forceswereevaluatedonlyeachsecondstepandaddedwiththe scalingfactor 2.  The molecular dynamics simulations were run in NPT ensemble at pH 7.4 and two temperatures (298 K and 310 K).  Trajectories were computed for 50 ns, the data were saved each 25 ps. Models building, structure refinement, molecular dynamics simulations, and analysis as well as the result presentation by using molecular graphics were performed by using the molecular modeling program YASARA Structure. Resultsanddiscussion. After equilibration the RMSD values for different systems are close to each other and change insignificantly (except Fe-containingHelicobactermustelaeurease), evidencing that their global structure is quite stable and that no significant  conformational transformations occur within these systems, whereas H.mustelaeurease structure revealed instability during the simulations. The presence of the competitive inhibitor in the active site did not change the Ni (1) and Ni (2) coordination numbers. There were observed no essential deformations of the geometry of the ions binding with the active site ligands as well. A similar situation was observed in the case of Mn-containingK. aerogenesurease too.  In the case of S. pasteurii urease apoenzyme, there were observed the insignificant shifts of the active site residues. The root mean square deviation of the residues of the active site of apoenzyme relative to holoenzyme was 0.65 Ǻ. In the absence of Ni ions in apoenzyme, the position of the inhibitor AHA within the active site is unstable and it gradually drifts and leaves the active site. For all ureases, the temperature increase from 298 K to 310 K had a little effect on the average distances “metal-ligand”. The temperature increase from 298 K to 310 K had an insignificant effect on the distances “metal-water” in the active site of the Ni-containing ureases S. pasteurii and K. aerogenes and a little influence on these distances in the Mn-containing K. aerogenes urease. The metallocentre structures in the Mn- and Ni-containing K. aerogenes ureases are very similar. Conclusions. There have been studied the structural role of the nickel ions in a urease active site, the influence of the temperature and the ion type on the structure of the urease active site. It have been shown that binding of the competitive inhibitor (acetohydroxamic acid, AHA) did not change the Ni ions coordination in the urease active site and  did not essentially effect the geometry of the active site near the nickel ions. The main factor of the inhibitor binding are the nickel ions. It have been shown that the active site structures of the Ni- and Mn-containing ureases KlebsiellaaerogenesandSporosarcinapasteurii are approximately identical. It have been shown that the metallocentre structure of these ureases are in general stable regardless of the urease source, the ion type and the temperature.