Structural and mechanistic studies of human chitinase

Chitinases are enzymes that hydrolyze chitin, a glucosamine polymer synthesized by lower organisms for structural purposes [1]. While humans do not synthetize chitin, they express two active chitinases, Chitotriosidase (hCHIT1) and Acidic Mammalian Chitinase (hAMCase). Both enzymes attracted attention due to their upregulation in immune system disorders [2,3]. They consist of a catalytic domain of 39 kDa and a chitin binding domain, joined by a hinge. The active site shows a cluster of three conserved acidic residues, E140, D138 and D136, linked by H-bonds, where D138 and E140 are involved in the hydrolysis reaction [1,3]. To increase our knowledge on the catalytic mechanism of human chitinases, we conducted a detailed structural analysis on hCHIT1. For this, we have improved the X-ray resolution of the apo hCHIT1 catalytic domain to 1Å. We investigated the protonation state on the catalytic site and detected a double conformation of D138, one in contact with D136 and a second one in contact with E140. Our analysis revealed for the first time different protonation states for each conformation of D138 (fig1). Interestingly, our X-ray data suggest that the catalytic E140, supposed to donate a proton in the catalytic reaction, is deprotonated in the apo form. To gain insight on the proton transition pathway during the hydrolysis, we have solved the X-ray structure of hCHIT1 complexed with the substrate at 1.05 Å. In comparison with the apo form, this structure shows a rearrangement of the protonation states of the catalytic triad triggered by the binding of the substrate. Our results led us to suggest a new hydrolysis model involving changes in the hydrogen bond network of the catalytic triad accompanied by a flip of D138 towards D136. This contributes to protonate E140, which then donates the proton to the substrate. To confirm the role of the active site's hydrogen network, we are currently studying CHIT1 by neutron crystallography and quantum mechanics.

X-ray structures of Aldose Reductase and AKR1B10 with the lead inhibitor JF0064

Human enzymes Aldose Reductase (AR or AKR1B1) and AKR1B10 are of biomedical interest because of their involvement in secondary diabetic complications and inflammatory disorders (AR) and in several type of cancers, e.g. hepatocellular carcinoma and smoking related lung cancer (AKR1B10). They belong to the Aldo-keto reductase family (AKRs) which present a highly conserved (α/β)8 barrel folding. Their substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable external loops. JF0064 (2,2',3,3',5,5',6,6'-octafluoro-4,4'-biphenyldiol) has been identified in a series of polyhalogenated compounds as a lead inhibitor of both enzymes. The determination of the X-ray structure of the AR:NADP+:JF0064 complex at ultrahigh-resolution (0.85 Å) allows to observe JF0064 interacting with the catalytic residue Tyr48 through a negatively charged hydroxyl group of the inhibitor (i.e., the acidic phenol). The non-competitive inhibition pattern observed for JF0064 with both enzymes suggests that this acidic hydroxyl group will also be present in the case of AKR1B10. The X-ray structure of the AKR1B10:NADP+:JF0064 complex (achieved with the combination of surface lysine methylation and the introduction of K125R/V301L mutations) and the comparison between both structures unveils some important hints for a structure-based drug design optimization.

UHR PX and NPC studies of H-FABP water network with tiny perdeuterated crystals

We have obtained very detailed information about the internal water molecules in the large internal cavity inside fatty acid binding (FABP) proteins , in the presence of bound fatty acids (FA), by Ultra High Resolution X-Ray Crystallography (UHR) to 0.7 Å and Neutron Protein Crystallography (NPC) to 1.9 Å using a "radically small" (V=0.05 mm3) crystal. These waters form a very well ordered dense cluster of 12 molecules, positioned between the hydrophilic internal wall of the cavity and the fatty acid molecule. This information has been used for a detailed electrostatic analysis based on the charge distribution description modeled in the multipole formalism and on the Atoms in Molecules theory. This information is also being used in molecular dynamics simulations of H-FABP and its complex with FA in order to quantify the energetic contribution of these internal waters to the binding energy. The experiment has been done with oleic acid, coming with the protein expressed in E. Coli. The results have been analyzed in order to understand the interactions between the FA, the internal water and the protein, and in particular the role played by the water molecules in determining the potency and specificity of FA binding to FABPs. The major tool for visualizing the water molecules inside the H-FABP cavity is UHR X-Ray Crystallography combined with NPC. UHR crystallographic structures give the positions of hydrogen and oxygen atoms for well-ordered water molecules. NPC determines hydrogen atom positions, particularly of water molecules which have multiple conformations, leading to the best possible crystallographic model. This model was then complemented by a transferred charge distribution to accurately determine the electrostatic and topological properties in the binding pocket, providing a description of the way water molecules in hydration layer contribute to the binding of ligand, which is essential to understand and model ligand binding.