Poisson-Boltzmann Calculations of Nonspecific Salt Effects on Protein-Protein Binding Free Energies

The salt dependence of the binding free energy of five protein-protein hetero-dimers and two homo-dimers/tetramers was calculated from numerical solutions to the Poisson-Boltzmann equation. Overall, the agreement with experimental values is very good. In all cases except one involving the highly charged lactoglobulin homo-dimer, increasing the salt concentration is found both experimentally and theoretically to decrease the binding affinity. To clarify the source of salt effects, the salt-dependent free energy of binding is partitioned into screening terms and to self-energy terms that involve the interaction of the charge distribution of a monomer with its own ion atmosphere. In six of the seven complexes studied, screening makes the largest contribution but self-energy effects can also be significant. The calculated salt effects are found to be insensitive to force-field parameters and to the internal dielectric constant assigned to the monomers. Nonlinearities due to high charge densities, which are extremely important in the binding of proteins to negatively charged membrane surfaces and to nucleic acids, make much smaller contributions to the protein-protein complexes studied here, with the exception of highly charged lactoglobulin dimers. Our results indicate that the Poisson-Boltzmann equation captures much of the physical basis of the nonspecific salt dependence of protein-protein complexation

A model of the complex between the PfEMP1 malaria protein and the human ICAM-1 receptor

Malaria is caused by protozoan parasites of the genus Plasmodium. Four species of Plasmodium can infect humans: R falciparum, P. malariae, P. vivax, and P. ovale. P. falciparum is the only able to cytoadhere to the surface of postcapillary endothelial cells. A key role in cytoadherence is played by the interaction between the PfEMP1 P. falciparum protein and the human intracellular adhesion molecule (ICAM-1) although very little is known about the molecular details of this complex. Here we propose a model for this interaction on the basis of a homology model of the functional domain of PfEMP1 and of the ICAM-1 three dimensional structures. Our model is consistent with the results of many experimental observations, provides a rational explanation for the different binding abilities of different strains of R falciparum and explains the reduced binding affinity of the A4 strain of P. falciparum for the ICAM-1(Kilifi) polymorphism. On the basis of our model, we can also explain why the murine ICAM-1, although sharing 70% sequence similarity with its human homologue, does not bind PfEMP1, and why the binding of fibrinogen and PfFMP1 to ICAM-1 is mutually exclusive. The model of the complex proposed here can serve as a useful tool for the design and interpretation of biochemical and immunological experimental results

Key Role of Proximal Water in Regulating Thermostable Proteins

Three homologous proteins with mesophilic, thermophilic and hyperthermophilic character have been studied via molecular dynamics simulations at four different temperatures in order to investigate how water controls thermostability. The water-exposed surface of the protein is shown to increase with the degree of thermophilicity, and the role of water in enhancing the protein internal flexibility and structural robustness is elucidated. The presence of water-water hydrogen bond clusters enveloping the macromolecule is shown to correlate with thermal robustness when going from the mesophilic to the hyperthermophilic variants. Our analysis indicates that essential contributions to thermostability stem from protein-water surface effects whereas the protein internal packing plays a minor role

Water around thermophilic proteins: the role of charged and apolar atoms

The thermal response of three proteins with mesophilic, thermophilic and hyperthermophilic character hints at the essential role played in thermostability by the protein-water interface. The formation of spanning water clusters enveloping the macromolecule and their resistance to thermal stress is shown to correlate with the charge distribution at the protein surface; in particular our findings suggest an effective role of the superficial charge distribution in stabilizing the global connectivity of the hydration water

From the Arctic to fetal life: physiological importance and structural basis of an 'additional' chloride-binding site in haemoglobin.

Haemoglobins from mammals of sub-Arctic and Arctic species, as well as fetal human Hb, are all characterized by a significantly lower Delta H of oxygenation compared with the majority of mammalian haemoglobins from temperate species (exceptions are represented by some cold-resistant species, such as cow, horse and pig). This has been interpreted as an adaptive mechanism of great importance from a physiological point of view. To date, the molecular basis of this thermodynamic characteristic is still not known. In the present study, we show that binding of extra chloride (with respect to adult human Hb) ions to Hb would significantly contribute to lowering the overall heat of oxygenation, thus providing a molecular basis for the low effect of temperature on the oxygenation-deoxygenation cycle. To this aim, the oxygen binding properties of bovine Hb, bear (Ursus arctos) Hb and horse Hb, which are representative of this series of haemoglobins, have been studied with special regard to the effect of heterotropic ligands, such as organic phosphates (namely 2,3-diphosphoglycerate) and chloride. Functional results are consistent with a mechanism for ligand binding that involves an additional binding site for chloride ion. Analysis of computational chemistry results, obtained by the GRID program, further confirm the hypothesis that the reason for the lower Delta H of oxygenation is mainly due to an increase in the number of the oxygen-linked chloride-binding sites

BurrH: a new modular DNA binding protein for genome engineering

The last few years have seen the increasing development of new DNA targeting molecular tools and strategies for precise genome editing. However, opportunities subsist to either improve or expand the current toolbox and further broaden the scope of possible biotechnological applications. Here we report the discovery and the characterization of BurrH, a new modular DNA binding protein from Burkholderia rhizoxinica that is composed of highly polymorphic DNA targeting modules. We also engineered this scaffold to create a new class of designer nucleases that can be used to efficiently induce in vivo targeted mutagenesis and targeted gene insertion at a desired locus

Identification by in silico and in vitro screenings of small organic molecules acting as reversible inhibitors of kallikreins

International audienceNetherton syndrome is caused by loss-of-function mutations in SPINK5 encoding the Kazal-type inhibitor LEKTI-1 leading to dysregulation of proteolytic cascades involving several kallikreins. We used both structure-based and ligand-based virtual screening computations to identify commercially available non-covalent inhibitors of human kallikrein 5 (hK5), a serine protease (trypsin-like) that plays a central role in the initiation of the molecular cascades leading to the Netherton syndrome phenotype. The efficacy and mechanism of inhibition of the identified new families of organic compounds were analyzed not only for hK5 but also on other proteases implicated in the cascades (hK7, hK14 and matriptase). These inhibitors are nontoxic on healthy human keratinocytes and are structurally different from traditional serine protease inhibitors validating their potential utility as initial hits to control proteolytic disorders observed in dermatological pathologies such as Netherton syndrome

BuD, a helix–loop–helix DNA-binding domain for genome modification

DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein-DNA interactions in protein scaffolds is key to providing 'toolkits' for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix-loop-helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin beta (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing