How To Design Selective Ligands for Highly Conserved Binding Sites: A Case Study Using N-Myristoyltransferases as a Model System

Under embargo until: 2020-08-19A model system of two related enzymes with conserved binding sites, namely N-myristoyltransferase from two different organisms, was studied to decipher the driving forces that lead to selective inhibition in such cases. Using a combination of computational and experimental tools, two different selectivity-determining features were identified. For some ligands, a change in side-chain flexibility appears to be responsible for selective inhibition. Remarkably, this was observed for residues orienting their side chains away from the ligands. For other ligands, selectivity is caused by interfering with a water molecule that binds more strongly to the off-target than to the target. On the basis of this finding, a virtual screen for selective compounds was conducted, resulting in three hit compounds with the desired selectivity profile. This study delivers a guideline on how to assess selectivity-determining features in proteins with conserved binding sites and to translate this knowledge into the design of selective inhibitors.acceptedVersio

Hemocyanin conformational changes associated with SDS-induced phenol oxidase activation

The enzymatic activity of phenoloxidase is assayed routinely in the presence of SDS. Similar assay conditions elicit phenoloxidase activity in another type 3 copper protein, namely hemocyanin, which normally functions as an oxygen carrier. The nature of the conformational changes induced in type 3 copper proteins by the denaturant SDS is unknown. This comparative study demonstrates that arthropod hemocyanins can be converted from being an oxygen carrier to a form which exhibits phenoloxidase activity by incubation with SDS, with accompanying changes in secondary and tertiary structure. Structural characterisation, using various biophysical methods, suggests that the micellar form of SDS is required to induce optimal conformational transitions in the protein which may result in opening a channel to the di-copper centre allowing bulky phenolic substrates access to the catalytic site

Crystallization and Preliminary Analysis of Crystals of the 24-Meric Hemocyanin of the Emperor Scorpion (Pandinus imperator)

Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (MW = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments

Structure of the Altitude Adapted Hemoglobin of Guinea Pig in the R2-State

Background: Guinea pigs are considered to be genetically adapted to a high altitude environment based on the consistent finding of a high oxygen affinity of their blood. Methodology/Principal Findings: The crystal structure of guinea pig hemoglobin at 1.8 A ˚ resolution suggests that the increased oxygen affinity of guinea pig hemoglobin can be explained by two factors, namely a decreased stability of the T-state and an increased stability of the R2-state. The destabilization of the T-state can be related to the substitution of a highly conserved proline (P44) to histidine (H44) in the a-subunit, which causes a steric hindrance with H97 of the b-subunit in the switch region. The stabilization of the R2-state is caused by two additional salt bridges at the b1/b2 interface. Conclusions/Significance: Both factors together are supposed to serve to shift the equilibrium between the conformational states towards the high affinity relaxed states resulting in an increased oxygen affinity

Tyrosinases from crustaceans form hexamers.

Tyrosinases, which are widely distributed among animals, plants and fungi, are involved in many biologically essential functions, including pigmentation, sclerotization, primary immune response and host defence. In the present study, we present a structural and physicochemical characterization of two new tyrosinases from the crustaceans Palinurus elephas (European spiny lobster) and Astacus leptodactylus (freshwater crayfish). In vivo, the purified crustacean tyrosinases occur as hexamers composed of one subunit type with a molecular mass of approx. 71 kDa. The tyrosinase hexamers appear to be similar to the haemocyanins, based on electron microscopy. Thus a careful purification protocol was developed to discriminate clearly between tyrosinases and the closely related haemocyanins. The physicochemical properties of haemocyanins and tyrosinases are different with respect to electronegativity and hydrophobicity. The hexameric nature of arthropod tyrosinases suggests that these proteins were the ideal predecessors from which to develop the oxygen-carrier protein haemocyanin, with its allosteric and co-operative properties, later on

Crystallization of the altitude adapted hemoglobin of guinea pig

Hemoglobin is the versatile oxygen carrier in the blood of vertebrates and a key factor for adaptation to live in high altitudes. Several structural changes are known to account for increased oxygen affinity in hemoglobin of altitude adapted animals such as llama and barheaded goose. Guinea pigs are adapted to live in high altitudes in the Andes and consequently their hemoglobin has an increased oxygen affinity. However, the structural changes responsible for the adaptation of guinea pig hemoglobin are unknown. Here we report the crystallization of guinea pig hemoglobin in the presence of 2.6 M ammonium sulfate and a preliminary analysis of the crystals. Crystals diffract up to a resolution of 2.0 A. They are orthorhombic with space group C 2 2 2(1) and cell dimensions a = 84.08 A, b = 90.21 A and c = 83.44 A