Elsa S. Henriques

On the modeling of snake venom serine proteinase interactions with benzamidine-based thrombin inhibitors

Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin‐like activities on fibrinogen and platelets, this being the case for the kinin‐releasing and fibrinogen‐clotting KN‐BJ from the venom of Bothrops jararaca. A three‐dimensional structural model of the KN‐BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV‐PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine‐based thrombin inhibitors α‐NAPAP, 3‐TAPAP, and 4‐TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN‐BJ2 toward those inhibitors.

In situ ultra-fast heat deposition does not perturb the structure of serum albumin

MnTPPS is a metallic water soluble porphyrin with high potential to be used as a contrast agent in photoacoustic tomography. In order to fully understand the interaction between MnTPPS and serum albumin and to investigate the effect of the light induced fast in situ heat deposition by MnTPPS in the protein, we performed several experimental studies using fluorescence and circular dichroism spectroscopies, as well as photoacoustic calorimetry. To identify the possible binding site(s) of the metalloporphyrin in serum albumin and to help interpret the spectroscopic results, a molecular docking exercise was also carried out. The fluorescence data indicate a 1 : 1 stoichiometry for the complex BSA : MnTPPS. The molecular docking results suggest one binding site at the subdomain IB of albumin, where Trp-134 is found, as the main binding site for MnTPPS. The CD data indicate no significant conformational changes of the BSA secondary structure upon MnTPPS binding and even after several minutes of laser excitation of MnTPPS. TR-PAC results show that the in situ heat deposition from MnTPPS does not cause any significant transient conformational change to the BSA structure. In conclusion, this work demonstrates that MnTPPS, in addition to the necessary physical and chemical properties to be used as a contrast agent in photoacoustic tomography, can be effectively carried by albumin and that in situ heat release following light absorption does not cause any significant damage to the protein structure.

A rational method for probing macromolecules dissociation: the antibody-hapten system

Abstract.The unbinding process of a protein-ligand complex of major biological interest was investigated by means of a computational approach at atomistic classical mechanical level. An energy minimisation-based technique was used to determine the dissociation paths of the system by probing only a relevant set of generalized coordinates. The complex problem was reduced to a low-dimensional scanning along a selected distance between the protein and the ligand. Orientational coordinates of the escaping fragment (the ligand) were also assessed in order to further characterise the unbinding. Solvent effects were accounted for by means of the Poisson–Boltzmann continuum model. The corresponding dissociation time was derived from the calculated barrier height, in compliance with the experimentally reported Arrhenius-like behaviour. The computed results are in good agreement with the available experimental data.

Modeling of the Toll-like receptor 3 and a putative Toll-like receptor 3 antagonist encoded by the African swine fever virus

African swine fever virus (ASFV) is a large double‐stranded DNA virus responsible for a lethal pig disease, to which no vaccine has ever been obtained. Its genome encodes a number of proteins involved in virus survival and transmission in its hosts, in particular proteins that inhibit signaling pathways in infected macrophages and, thus, interfere with the hosts innate immune response. A recently identified novel ASFV viral protein (pI329L) was found to inhibit the Toll‐like receptor 3 (TLR3) signaling pathway, TLR3 being a crucial “danger detector.” pI329L has been predicted to be a transmembrane protein containing extracellular putative leucine‐rich repeats similar to TLR3, suggesting that pI329L might act as a TLR3 decoy. To explore this idea, we used comparative modeling and other structure prediction protocols to propose (a) a model for the TLR3–Toll‐interleukin‐1 receptor homodimer and (b) a structural fold for pI329L, detailed at atomistic level for its cytoplasmic domain. As this later domain shares only remote sequence relationships with the available TLR3 templates, a more complex modeling strategy was employed that combines the iterative implementation of (multi)threading/assembly/refinement (I‐TASSER) structural prediction with expertise‐guided posterior refinement. The final pI329L model presents a plausible fold, good structural quality, is consistent with the available experimental data, and it corroborates our hypothesis of pI329L being a TLR3 antagonist.

The search for a new model structure of beta-factor XIIa.

We present the search for a new model of β-factor XIIa, a blood coagulation enzyme, with an unknown experimental 3D-structure. We decided to build not one but three different models using different homologous proteins as well as different techniques and different modellers. Additional studies, including extensive molecular dynamics simulations on the solvated state, allowed us to draw several conclusions concerning homology modelling, in general, and β-factor XIIa, in particular.

MODELLING ENZYME - LIGAND INTERACTIONS

Publisher Summary The purpose of this chapter is to discuss enzyme-ligand interactions. Most physiological and pharmacological responses are mediated by specific receptor-ligand interactions. These receptors are macromolecules specialized in recognizing a specific molecular pattern from the large number of surrounding molecular species with which they could interact, the term “receptor” being used to designate pharmacological receptors, enzymes, antibodies and DNA. The major aspects that determine a biomolecular recognition event, all of them involving changes in both entropy and enthalpy, are: (1) the structural and energetic complementarity between the ligand and the receptor, (2) the conformational rearrangements both structures undergo upon complexation, (3) their desolvation, and (4) the loss of rotational and translational freedom of the ligand when it binds to the active site. The difficulty lies in quantitate and scale the relative contributions of those aspects to the free energy of binding (hence, predicting the affinity).