However, this protein failed to crystallize

However, this protein failed to crystallize. in LPS synthesis, and previous structures revealed that LpxH PU-WS13 has a helical cap that binds its lipid substrates. Here, crystallography and hydrogenCdeuterium exchange MS provided evidence for a highly flexible substrate-binding cap in LpxH. Furthermore, molecular dynamics simulations disclosed how the helices of the cap may open to allow substrate entry. The predicted opening mechanism was supported by activity assays of LpxH variants. Finally, we confirmed biochemically that LpxH is inhibited by a previously identified antibacterial compound, determined the potency of this inhibitor, and modeled its binding mode in the LpxH active site. In summary, our work provides evidence that PU-WS13 the substrate-binding cap of LpxH is highly dynamic, thus allowing for facile substrate binding and product release between the capping helices. Our results also pave the way for the rational design of more potent LpxH inhibitors. and bound to the product (lipid X) and in the DKK1 unbound form (14, 15). LpxH is a hydrolase that catalyzes the cleavage of the phosphoanhydride of UDP-2,3-diacylglucosamine (UDPCDAG) utilizing a dimanganese center (8, 16). Approximately 70% of Gram-negative bacteria utilize LpxH, whereas utilize the distantly related LpxG, and most of the rest utilize the nonhomologous enzyme LpxI (17, 18). LpxH has a calcineurin-like metal-dependent phosphoesterase fold with the addition of a unique helical cap, comprising three -helices, that covers the active site and binds the lipid substrates (14, 15). Although previous structures suggested that the capping domain is stably attached to the rest of the protein and only becomes slightly disordered in the absence of ligand (15), we provide multiple lines of evidence that the capping domain is highly dynamic in the absence of ligand. These results have implications for the mechanism of substrate binding and product release: although slight disordering of the third capping helix, as observed previously, could allow lipids to enter and exit the active site, a more extensive conformational change would enable facile binding and release of lipids through a wider opening between the capping helices. Results and discussion Crystal structure A solubilized version of LpxH including four solubilizing mutations (F141H, L142S, L146S, and F147H) and four surface entropy reduction mutations (E14A, E15A, K161T, and E162A) (LpxH4+4) was generated to improve protein expression and crystallization. LpxH4+4 crystallized in plate-shaped crystals that diffracted to 2.00 ?, and the structure was solved by molecular replacement (Table 1) (PDB code 5WLY). The core phosphoesterase domain of LpxH is composed of two 5-stranded -sheets sandwiched together by -helices, and this domain overlays very well with the previous LpxH structures (14, 15, 19) (Figs. 1and ?and22LpxH were displaced from the position observed in previous structures and were largely disordered (Fig. 2LpxH (PaLpxH) did show increased disorder and an altered conformation where the PU-WS13 capping helices bind the product head group and connect to the core domain, all of those other helices remained loaded together with the energetic site (Fig. 2LpxH, the helices are detached in the energetic site, in support of the middle part (residues 131C161) is seen. This conformation is normally fortuitously stabilized by crystal connections (Fig. S1) and is probable transient in alternative. However, this framework shows that the capping helices are a lot more versatile and disordered within the lack of substrate than was obvious from prior LpxH buildings. This framework of LpxH was the impetus for even more experiments to look at the movement from the cover. You should remember that the capping helices of LpxH4+4, which includes six mutations inside the capping helices, or of LpxH generally could be more flexible PU-WS13 than those in or LpxH inherently. Alternatively, in prior LpxH crystal buildings, the shut conformation was stabilized by binding to lipid X and/or crystal connections wherein the capping domains had been often loaded against one another, which may describe the limited motion seen in the apo PaLpxH framework (14, 15). As the buildings and sequences of LpxH from these types are highly very similar (Fig. 2 and.