Técnica Quirúrgica de reducción con cerclaje y enclavado

Potential Binding Sites on LlaHisZG

Three distinct histidine binding sites have been proposed for LlaHisZGS. The first (designated site one) is located in a region highly conserved between HisZ and histidyl-tRNA synthetase. Champagne et al. used mutagenesis to attribute histidine binding to three conserved residues at this site (Glu130, Tyr268 and Tyr269).20 _{Whether these} represent the histidine binding residues, or if mutation of these residues disrupted the allosteric signal transduction pathway, is yet to be deduced. Sites two and three reside at the interface between LlaHisZ and LlaHisGS and were proposed based on comparison with the histidine-bound crystal structure of TmaHisZGS (PDB 1USY).19 Induced fit docking of histidine into each of the three potential binding sites on the LlaHisZGS complex was performed by Dr Wanting Jiao, with the intention of identifying the biologically relevant histidine binding sites(s). An ensemble of histidine poses was generated and Prime structure prediction was used to accommodate the ligand by reorienting side chains in the proposed binding site. The ligand was then re-docked into the lowest energy protein structure and poses were ranked by their Glide docking score. The Glide score is an empirical measure that approximates the free energy of ligand binding.76,77 Analysis of these models is presented in this section.

2.6.1 Histidine Docking into Site One of LlaHisZGS

Preparation of the LlaHisZGS complex for induced fit docking resulted in Glu130 being assigned to a neutral state, instead of the negatively charged state expected of this residue. It is unclear why this occurred, therefore the induced fit docking of histidine into site one was evaluated under both negative and neutral charge states. The top scored pose of histidine docked into site one with a neutral Glu130 has a Glide docking score of −6.009. This pose reveals no significant interaction between histidine and the histidine-binding residues proposed by Champagne et al.20 _{(Figure 2.19). The Tyr268 and Tyr269} side chains both orient away from the bound histidine. Glu130 lies in close proximity to histidine, however there is no evidence to suggest that this residue is involved in stabilisation of the ligand in the binding pocket. This pose highlights Lys265, Asp84, and Tyr291 as potential histidine binding residues. The carboxylate group of histidine forms both a hydrogen bond and a salt bridge with the Lys265 side chain. A salt bridge is also formed between the amino group of histidine and the side chain of Asp84 and pi-pi stacking interactions are established with the side chain of Tyr291.

Figure 2.19 The highest scored docking pose of histidine in site one of LlaHisZGS with a neutral Glu130.

The proposed histidine binding residues determined by mutagenesis (yellow sticks) show no interaction with the histidine ligand (green). Grey sticks are used to show the histidine binding residues in this pose. The cartoon structure of LlaHisZ is coloured red.

When histidine was docked into site one with a negative Glu130, the highest scored pose had a docking score of −6.041. Histidine adopts a different conformation in this pose, compared to the previously described pose, which suggests that this site is sufficiently spacious to accommodate histidine binding in different orientations (Figure 2.20). The imidazole ring of histidine forms pi-pi stacking interactions with Tyr291 and the side chain of Asp84 forms a salt bridge with the histidine amino group, as described previously. The carboxylate group of histidine also interacts with the side chains of Lys265 and Gln126. The three proposed histidine binding residues again show no interaction with the ligand.

Figure 2.20 The highest scored docking pose of histidine docked into site one with a negative Glu130. LlaHisZ is coloured red, the previously proposed histidine binding residues are shown in yellow, and grey sticks represent the histidine binding residues.

2.6.2 Histidine Docking into Site Two of LlaHisZGS

Docking of histidine into potential binding site two of LlaHisZGS, at the interface of LlaHisZ and LlaHisGS, resulted in a pose with a docking score of -8.071 (Figure 2.21). Hydrogen bonding is observed between the carboxylate group of histidine and the side chains of Lys213 and Asn196 from LlaHisZ. The backbone amino group of Lys213 also appears to stabilise the imidazole group of histidine by hydrogen bonding. The amino group of histidine forms salt bridges with Glu200 and Glu203 from LlaHisGS.

Figure 2.21 Histidine (green) docked into potential binding site two at the interface of LlaHisGS (blue) and

LlaHisZ (red). The histidine binding residues are coloured grey.

2.6.3 Histidine Docking into Site Three of LlaHisZGS

Docking of histidine into potential binding site three of LlaHisZGS, also located at the LlaHisZ−LlaHisGS interface, resulted in a pose with a docking score of -5.813 (Figure 2.22). In this pose, the amino group and the imidazole group of histidine establish hydrogen bonds with the side chain of Asp191 from LlaHisZ. The Glu83 and Lys70 of LlaHisGS also form salt bridges with the carboxylate group of histidine.

Figure 2.22 Histidine (green) docked into potential binding site three at the interface of LlaHisGS (blue)

2.6.4 Where Does Histidine Bind?

Induced fit docking of histidine into potential binding site two, at the interface of LlaHisZ and LlaHisGS, gave the best (most negative) docking score. This binding site, along with potential binding site three, was identified by comparison with the histidine-bound TmaHisZGS crystal structure. It is tempting to conclude that this is the functional allosteric site and that the Glu130, Tyr268 and Tyr269 residues of site one are critical for allosteric signal transmission, but not involved in ligand binding. Induced fit docking, however, was performed on the unliganded LlaHisZGS structure and binding of histidine has been shown to trigger a conformational change of the protein. The algorithm used for induced-fit docking compensates for changes in side chain positioning but does not account for the large conformational changes observed by SAXS.