Developing an energy landscape for the novel function of a (beta/alpha)8 barrel: ammonia conduction through HisF

Abstract

HisH-hisF is a multidomain globular protein complex; hisH is a class I glutamine amidotransferase that hydrolyzes glutamine to form ammonia, and hisF is a (beta/alpha)8 barrel cyclase that completes the ring formation of imidizole glycerol phosphate synthase. Together, hisH and hisF form a glutamine amidotransferase that carries out the fifth step of the histidine biosynthetic pathway. Recently, it has been suggested that the (beta/alpha)8 barrel participates in a novel function: to channel ammonia from the active site of hisH to the active site of hisF. The present study presents a series of molecular dynamic simulations that investigate the channeling function of hisF. This article reconstructs potentials of mean force for the conduction of ammonia through the channel, and the entrance of ammonia through the strictly conserved channel gate, in both a closed and a hypothetical open conformation. The resulting energy landscape within the channel supports the idea that ammonia does indeed pass through the barrel, interacting with conserved hydrophilic residues along the way. The proposed open conformation, which involves an alternate rotamer state of one of the gate residues, presents only an approximately 2.5-kcal energy barrier to ammonia entry. Another alternate open-gate conformation, which may play a role in non-nitrogen-fixing organisms, is deduced through bioinformatics.

Fig. 1.

HisH–hisF complex. Snapshot from one of the trajectories is shown, with NH₃ within the barrel of hisF. Secondary structure motifs, hisH active site, strictly conserved Tyr-138 used in proposed open-gate conformation, and conserved residues in the hisF barrel are shown explicitly. NH₃ is shown in van der Waals representation.

Fig. 2.

Multiple sequence alignment: all three domains of life are shown as an excerpt from an alignment performed with 54 available sequences (yeast, Saccharomyces cerevisiae; THEMA, T. maritima; METTH, Methanobacterium thermoautotrophicum) BUCAP (Buchnera aphidicola) was chosen to represent nitrogen-fixing organisms. Strictly conserved residues across all sequences are shown in boldface, and residues corresponding to known and putative gating residues are marked with an asterisk. Numbering corresponds to THEMA. Note the strict conservation of gating residues Arg-5, Glu-46, Lys-99, and Glu-167. Aspartate residues suggested in possible gating mechanism (Asp-98, Asp-219) are conserved among non-nitrogen fixing organisms.

Fig. 3.

(a) Closed gate is present in five of six crystal structures, gate diameter 2.8 Å. (b) Chain C of 1GPW.pdb, partially closed, gate diameter 3.2 Å. (c) Top view of proposed open gate, Lys-99 within hydrogen-bonding distance of Tyr-138 of hisH, gate diameter 5.8 Å. (d) Side view of proposed open gate, 2.7-Å hydrogen bond indicated by gray line.

Fig. 4.

PMF reconstruction. (a) Force in kcal/mol per Ų for five trajectories through the closed channel. (b) PMF shown for (i) closed- and (ii) open-gate conformations.

Fig. 5.

Dipole orientation parameter of ammonia averaged over all eight open-gate trajectories shows a clear directional preference of ammonia at different positions in the channel corresponding to conserved hydrophilic residues in the channel. θ is the angle between the channel axis (z axis) and the normalized ammonia dipole vector (3).