Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation.

Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (k(off)). Since no techniques are available to estimate k(off), we report herewith a method to determine k(off) based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k(off) value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)-cyanide) was determined at pH 9.2 and 20.0 degrees C. Mixing Mb(III)-cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)-cyanide with the concomitant formation of ferrous nitrosylated Mb. Since kinetics of reductive nitrosylation of Mb(III) is much faster than Mb(III)-cyanide dissociation, the k(off) value, representing the rate-limiting step, can be directly determined. The k(off) value obtained experimentally matches very well to that calculated from values of the second-order rate constant (k(on)) and of the dissociation equilibrium constant (K) for cyanide binding to Mb(III) (k(off)=k(on)xK).

Structural basis for resistance of the genotype 2b hepatitis C virus NS5B polymerase to site A non-nucleoside inhibitors.

Hepatitis C virus (HCV) exists in six major genotypes. Compared with the 1b enzyme, genotype 2b HCV polymerase exhibits a more than 100-fold reduction in sensitivity to the indole-N-acetamide class of non-nucleoside inhibitors. These compounds have been shown to bind in a pocket occupied by helix A of the mobile Lambda1 loop in the apoenzyme. The three-dimensional structure of the HCV polymerase from genotype 2b was determined to 1.9-A resolution and compared with the genotype 1b enzyme. This structural analysis suggests that genotypic variants result in a different shape of the inhibitor binding site. Mutants of the inhibitor binding pocket were generated in a 1b enzyme and evaluated for their binding affinity and sensitivity to inhibition by indole-N-acetamides. Most of the point mutants showed little variation in activity and IC(50), with the exception of 15- and 7-fold increases in IC(50) for Leu392Ile and Val494Ala mutants (1b-->2b), respectively. Furthermore, a 1b replicon with 20-fold resistance to this class of inhibitors was selected and shown to contain the Leu392Ile mutation. Chimeric enzymes, where the 2b fingertip Lambda1 loop, pocket or both replaced the corresponding regions of the 1b enzyme, were also generated. The fingertip chimera retained 1b-like inhibitor binding affinity, whereas the other two chimeric constructs and the 2b enzyme displayed between 50- and 100-fold reduction in binding affinity. Together, these data suggest that differences in the amino acid composition and shape of the indole-N-acetamide binding pocket are responsible for the resistance of the 2b polymerase to this class of inhibitors.

Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial hypercholesterolemia mutants.

PCSK9 regulates low density lipoprotein receptor (LDLR) levels and consequently is a target for the prevention of atherosclerosis and coronary heart disease. Here we studied the interaction, of LDLR EGF(A/AB) repeats with PCSK9. We show that PCSK9 binds the EGF(AB) repeats in a pH-dependent manner. Although the PCSK9 C-terminal domain is not involved in LDLR binding, PCSK9 autocleavage is required. Moreover, we report the x-ray structure of the PCSK9DeltaC-EGF(AB) complex at neutral pH. Compared with the low pH PCSK9-EGF(A) structure, the new structure revealed rearrangement of the EGF(A) His-306 side chain and disruption of the salt bridge with PCSK9 Asp-374, thus suggesting the basis for enhanced interaction at low pH. In addition, the structure of PCSK9DeltaC bound to EGF(AB)(H306Y), a mutant associated with familial hypercholesterolemia (FH), reveals that the Tyr-306 side chain forms a hydrogen bond with PCSK9 Asp-374, thus mimicking His-306 in the low pH conformation. Consistently, Tyr-306 confers increased affinity for PCSK9. Importantly, we found that although the EGF(AB)(H306Y)-PCSK9 interaction is pH-independent, LDLR(H306Y) binds PCSK9 50-fold better at low pH, suggesting that factors other than His-306 contribute to the pH dependence of PCSK9-LDLR binding. Further, we determined the structures of EGF(AB) bound to PCSK9DeltaC containing the FH-associated D374Y and D374H mutations, revealing additional interactions with EGF(A) mediated by Tyr-374/His-374 and providing a rationale for their disease phenotypes. Finally, we report the inhibitory properties of EGF repeats in a cellular assay measuring LDL uptake.

Development and optimization of a binding assay for histone deacetylase 4 using surface plasmon resonance.

Histone deacetylase 4 (HDAC4) is a histone deacetylase profoundly involved in cell differentiation and in the pathogenesis of cancer. The histone deacetylase inhibitors are a new, promising class of anticancer agents. The screening of molecular interactions involving determination of the affinity of drug candidates is an integral part of the drug discovery process. Here we report the development of an assay using surface plasmon resonance for the analysis of HDAC4-small molecule interactions. We describe a new cloning and purification strategy that can be used to set up surface plasmon resonance experiments with other recombinant proteins.

Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8-substrate complex.

Histone deacetylases (HDACs)-an enzyme family that deacetylates histones and non-histone proteins-are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 A resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor-HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.

Effects of pH and low density lipoprotein (LDL) on PCSK9-dependent LDL receptor regulation.

Mutations within PCSK9 (proprotein convertase subtilisin/kexin type 9) are associated with dominant forms of familial hyper- and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an approximately 150-fold higher affinity at an acidic endosomal pH (K(D) = 4.19 nm) compared with a neutral pH (K(D) = 628 nm). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.

Structural basis for HIV-1 neutralization by a gp41 fusion intermediate-directed antibody.

Elicitation of potent and broadly neutralizing antibodies is an important goal in designing an effective human immunodeficiency virus-1 (HIV-1) vaccine. The HIV-1 gp41 inner-core trimer represents a functionally and structurally conserved target for therapeutics. Here we report the 2.0-A-resolution crystal structure of the complex between the antigen-binding fragment of D5, an HIV-1 cross-neutralizing antibody, and 5-helix, a gp41 inner-core mimetic. Both binding and neutralization depend on residues in the D5 CDR H2 loop protruding into the conserved gp41 hydrophobic pocket, as well as a large pocket in D5 surrounding core gp41 residues. Kinetic analysis of D5 mutants with perturbed D5-gp41 interactions suggests that D5 persistence at the fusion intermediate is crucial for neutralization. Thus, our data validate the gp41 N-peptide trimer fusion intermediate as a target for neutralizing antibodies and provide a template for identification of more potent and broadly neutralizing molecules.

A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope.

HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H/I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro. D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusion-inhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a "hot spot" for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.

Nitrosylation of rabbit ferrous heme-hemopexin.

Hemopexin (HPX) serves as a trap for toxic plasma heme, ensuring its complete clearance by transportation to the liver. Moreover, HPX-heme has been postulated to play a key role in the homeostasis of nitric oxide (NO). Here, the thermodynamics for NO binding to rabbit ferrous HPX-heme as well as the EPR and optical absorption spectroscopic properties of rabbit ferrous nitrosylated HPX-heme (HPX-heme-NO) are reported. The value of the dissociation equilibrium constant for NO binding to rabbit ferrous HPX-heme (i.e., H) is (1.4+/-0.2)x10(-7) M, at pH 7.0 and 10.0 degrees C; the value of H is unaffected by sodium chloride. At pH 7.0, rabbit ferrous HPX-heme-NO is a six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry and by epsilon=146 mM(-1) cm(-1) at 419 nm. At pH 4.0, rabbit ferrous HPX-heme-NO is a five-coordinate heme-iron species, characterized by an X-band EPR spectrum with three-line splitting centered at 334 mT and by epsilon=74 mM(-1) cm(-1) at 387 nm. The p K(a) value of the reversible pH-induced six- to five-coordinate spectroscopic transition is 4.8+/-0.1 in the absence of sodium chloride and 4.3+/-0.1 in the presence of 1.5x10(-1) M sodium chloride. This result is in agreement with the effect of sodium chloride on rabbit HPX-heme stability. The present data have been analyzed in parallel with those of a related heme model compound and heme-protein systems.

Cyanide binding to truncated hemoglobins: a crystallographic and kinetic study.

Cyanide is one of the few diatomic ligands able to interact with the ferric and ferrous heme-Fe atom. Here, the X-ray crystal structure of the cyanide derivative of ferric Mycobacterium tuberculosis truncated hemoglobin-N (M. tuberculosis trHbN) has been determined at 2.0 A (R-general = 17.8% and R-free = 23.5%), and analyzed in parallel with those of M. tuberculosis truncated hemoglobin-O (M. tuberculosis trHbO), Chlamydomonas eugametos truncated hemoglobin (C. eugametos trHb), and sperm whale myoglobin, generally taken as a molecular model. Cyanide binding to M. tuberculosis trHbN is stabilized directly by residue TyrB10(33), which may assist the deprotonation of the incoming ligand and the protonation of the outcoming cyanide. In M. tuberculosis trHbO and in C. eugametos trHb the ligand is stabilized by the distal pocket residues TyrCD1(36) and TrpG8(88), and by the TyrB10(20) - GlnE7(41) - GlnE11(45) triad, respectively. Moreover, kinetics for cyanide binding to ferric M. tuberculosis trHbN and trHbO and C. eugametos trHb, for ligand dissociation from the ferrous trHbs, and for the reduction of the heme-Fe(III)-cyanide complex have been determined, at pH 7.0 and 20.0 degrees C. Despite the different heme distal site structures and ligand interactions, values of the rate constant for cyanide binding to ferric (non)vertebrate heme proteins are similar, being influenced mainly by the presence in the heme pocket of proton acceptor group(s), whose function is to assist the deprotonation of the incoming ligand (i.e., HCN). On the other hand, values of the rate constant for the reduction of the heme-Fe(III)-cyanide (non)vertebrate globins span over several orders of magnitude, reflecting the different ability of the heme proteins considered to give productive complex(es) with dithionite or its reducing species SO(2)(-). Furthermore, values of the rate constant for ligand dissociation from heme-Fe(II)-cyanide (non)vertebrate heme proteins are very different, reflecting the different nature and geometry of the heme distal residue(s) hydrogen-bonded to the heme-bound cyanide.

Determination of ferric heme-human serum albumin by 1H NMR relaxometry.

A new method for the accurate determination of ferric heme-human serum albumin (heme-HSA) at concentrations down to the physiological level, i.e., in the micromolar concentration range, is proposed. This method is based on the (1)H NMR relaxometric properties of heme-HSA. Actually, the binding of the paramagnetic ferric heme to the primary binding site of HSA determines a strong paramagnetic enhancement of the water (1)H NMR relaxation rate. Although a linear relationship may be seen by operating at 20 MHz on conventional electromagnets, the method here reported is improved by working at 0.02 MHz on a field-cycling instrument. This (1)H NMR relaxometric method does not suffer from the presence in serum of heme catabolites (e.g., bilirubin) that affect significantly the optical determination of ferric heme-HSA in the micromolar concentration range. Paramagnetic ferric hemoglobin contribution may be selectively quenched by cyanide binding.

The heme-iron geometry of ferrous nitrosylated heme-serum lipoproteins, hemopexin, and albumin: a comparative EPR study.

Serum high and low density lipoproteins, albumin, and hemopexin (HDL, LDL, SA, and HPX, respectively) serve as traps of toxic plasma heme and participate in its complete clearance by transportation to the liver. Moreover, SA-(heme) and HPX-heme have been proposed to facilitate NO scavenging in vivo. Here, the EPR-spectroscopic properties of ferrous nitrosylated heme-human high and low density lipoproteins (HDL-heme-NO and LDL-heme-NO, respectively) as well as of ferrous nitrosylated heme-rabbit serum hemopexin (HPX-heme-NO) are reported and analyzed in parallel with those of ferrous nitrosylated heme-human serum albumin (SA-heme-NO). HDL-heme-NO and LDL-heme-NO as well as SA-heme-NO, in the absence of allosteric effectors (i.e., N-form), are five-coordinate heme-iron species, characterized by the three-line splitting observed in the high magnetic field region of the X-band EPR spectrum. On the other hand, SA-heme-NO, in the presence of drugs (i.e., B-form), and HPX-heme-NO are six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry. The heme-iron coordination state of HDL-heme-NO, LDL-heme-NO, SA-heme-NO, and HPX-heme-NO is in keeping with values of ferric heme dissociation rate constants which decrease in the following order: LDL>HDL>SA>HPX. Altogether, these observations suggest that HPX displays a cleft much more suitable for heme binding than other heme-carriers.

Molecular bases for the anti-parasitic effect of NO (Review).

Nitric oxide (NO) has emerged as an important cytotoxic and cytostatic effector for a number of pathogens, including viruses, bacteria, fungi, and parasites. When the microbicidal effect of NO occurs, the NO-mediated S-nitrosylation of cysteine containing proteins (e.g., cysteine proteases) appears to be a common and widespread mechanism. This overview concerns parasitic cysteine proteases as NO targets, providing molecular bases for the parasiticidal effect of NO.