Multiple drugs and multiple targets: An analysis of the electrostatic determinants of binding between non‐nucleoside HIV‐1 reverse transcriptase inhibitors and variants of HIV‐1 RT

We present a systematic, computational analysis of the electrostatic component of binding of three HIV‐1 RT inhibitors—nevirapine (NVP), efavirenz (EFV), and the recently approved rilpivirine (RPV)—to wild‐type (WT) and mutant variants of RT. Electrostatic charge optimization was applied to determine how suited each molecule's charge distribution is for binding WT and individual mutants of HIV‐1 RT. Although the charge distributions of NVP and EFV are rather far from being optimal for tight binding, RPVs charge distribution is close to the theoretical, optimal charge distribution for binding WT HIV‐1 RT, although slight changes in charge can dramatically impact binding energetics. Moreover, toward the L100I/K103N double mutant, RPVs charge distribution is quite far from optimal. We also determine the contributions of chemical moieties on each molecule toward the electrostatic component of binding and show that different regions of a drug molecule may be used for recognition by different RT variants. The electrostatic contributions of certain RT residues toward drug binding are also computed to highlight critical residues for each interaction. Finally, the charge distribution of RPV is optimized to promiscuously bind to three RT variants rather than to each one in turn, with the resulting charge distribution being a compromise between the optimal charge distributions to each individual variant. Taken together, this work demonstrates that even in a binding site considered quite hydrophobic, electrostatics play a subtle yet varying role that must be considered in designing next‐generation molecules that recognize rapidly mutating targets. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Structure of HIV‐1 reverse transcriptase/d4TTP complex: Novel DNA cross‐linking site and pH‐dependent conformational changes

Stavudine (d4T, 2′,3′‐didehydro‐2′,3′‐dideoxythymidine) was one of the first chain‐terminating nucleoside analogs used to treat HIV infection. We present the first structure of the active, triphosphate form of d4T (d4TTP) bound to a catalytic complex of HIV‐1 RT/dsDNA template‐primer. We also present a new strategy for disulfide (S–S) chemical cross‐linking between N⁶ of a modified adenine at the second overhang base to I63C in the fingers subdomain of RT. The cross‐link site is upstream of the duplex‐binding region of RT, however, the structure is very similar to published RT structures with cross‐linking to Q258C in the thumb, which suggests that cross‐linking at either site does not appreciably perturb the RT/DNA structures. RT has a catalytic maximum at pH 7.5. We determined the X‐ray structures of the I63C‐RT/dsDNA/d4TTP cross‐linked complexes at pH 7, 7.5, 8, 8.5, 9, and 9.5. We found small (~0.5 Å), pH‐dependent motions of the fingers subdomain that folds in to form the dNTP‐binding pocket. We propose that the pH‐activity profile of RT relates to this motion of the fingers. Due to side effects of neuropathy and lipodystrophy, use of d4T has been stopped in most countries, however, chemical modification of d4T might lead to the development of a new class of nucleoside analogs targeting RNA and DNA polymerases.     

Structural perspectives on HIV‐1 Vif and APOBEC3 restriction factor interactions

Human immunodeficiency virus (HIV) is a retroviral pathogen that targets human immune cells such as CD4⁺ T cells, macrophages, and dendritic cells. The human apo lipoprotein B mRNA‐ e diting c atalytic polypeptide 3 (APOBEC3 or A3) cytidine deaminases are a key class of intrinsic restriction factors that inhibit replication of HIV. When HIV‐1 enters the cell, the immune system responds by inducing the activation of the A3 family proteins, which convert cytosines to uracils in single‐stranded DNA replication intermediates, neutralizing the virus. HIV counteracts this intrinsic immune response by encoding a protein termed viral infectivity factor (Vif). Vif targets A3 to an E3 ubiquitin ligase complex for poly‐ubiquitination and proteasomal degradation. Vif is unique in that it can recognize and counteract multiple A3 restriction factor substrates. Structural biology studies have provided significant insights into the overall architectures and functions of Vif and A3 proteins; however, a structure of the Vif‐A3 complex has remained elusive. In this review, we summarize and reanalyze experimental data from recent structural, biochemical, and functional studies to provide key perspectives on the residues involved in Vif‐A3 protein–protein interactions.     

Pathway mapping and development of disease‐specific biomarkers: protein‐based network biomarkers

It is known that a disease is rarely a consequence of an abnormality of a single gene, but reflects the interactions of various processes in a complex network. Annotated molecular networks offer new opportunities to understand diseases within a systems biology framework and provide an excellent substrate for network‐based identification of biomarkers. The network biomarkers and dynamic network biomarkers (DNBs) represent new types of biomarkers with protein–protein or gene–gene interactions that can be monitored and evaluated at different stages and time‐points during development of disease. Clinical bioinformatics as a new way to combine clinical measurements and signs with human tissue‐generated bioinformatics is crucial to translate biomarkers into clinical application, validate the disease specificity, and understand the role of biomarkers in clinical settings. In this article, the recent advances and developments on network biomarkers and DNBs are comprehensively reviewed. How network biomarkers help a better understanding of molecular mechanism of diseases, the advantages and constraints of network biomarkers for clinical application, clinical bioinformatics as a bridge to the development of diseases‐specific, stage‐specific, severity‐specific and therapy predictive biomarkers, and the potentials of network biomarkers are also discussed.     

Target of rapamycin FATC domain as a general membrane anchor: The FKBP‐12 like domain of FKBP38 as a case study

Increased efforts have been undertaken to better understand the formation of signaling complexes at cellular membranes. Since the preparation of proteins containing a transmembrane domain or a prenylation motif is generally challenging an alternative membrane anchoring unit that is easy to attach, water‐soluble and binds to different membrane mimetics would find broad application. The 33‐residue long FATC domain of yeast TOR1 (y1fatc) fulfills these criteria and binds to neutral and negatively charged micelles, bicelles, and liposomes. As a case study, we fused it to the FKBP506‐binding region of the protein FKBP38 (FKBP38‐BD) and used ¹H–¹⁵N NMR spectroscopy to characterize localization of the chimeric protein to micelles, bicelles, and liposomes. Based on these and published data for y1fatc, its use as a C‐terminally attachable membrane anchor for other proteins is compatible with a wide range of buffer conditions (pH circa 6–8.5, NaCl 0 to >150 mM, presence of reducing agents, different salts such as MgCl₂ and CaCl₂). The high water‐solubility of y1fatc enables its use for titration experiments against a membrane‐localized interaction partner of the fused target protein. Results from studies with peptides corresponding to the C‐terminal 17–11 residues of the 33‐residue long domain by 1D ¹H NMR and CD spectroscopy indicate that they still can interact with membrane mimetics. Thus, they may be used as membrane anchors if the full y1fatc sequence is disturbing or if a chemically synthesized y1fatc peptide shall be attached by native chemical ligation, for example, unlabeled peptide to ¹⁵N‐labeled target protein for NMR studies.     

Roles of conserved tryptophans in trimerization of HIV‐1 membrane‐proximal external regions: Implications for virucidal design via alchemical free‐energy molecular simulations

The Dual‐Action Virolytic Entry Inhibitors, or “DAVEI's,” are a class of recombinant fusions of a lectin, a linker polypeptide, and a 15‐residue fragment from the membrane‐proximal external region (MPER) of HIV‐1 gp41. DAVEI's trigger rupture of HIV‐1 virions, and the interaction site between DAVEI MPER and HIV‐1 lies in the gp41 component of the envelope glycoprotein Env. Here, we explore the hypothesis that DAVEI MPER engages Env gp41 in a mode structurally similar to a crystallographic MPER trimer. We used alchemical free‐energy perturbation to assess the thermodynamic roles of each of the four conserved tryptophan residues on each protomer of MPER₃. We found that a W666A mutation had a large positive for all three protomers, while W672A had a large positive for only two of the three protomers, with the other tryptophans remaining unimportant contributors to MPER₃ stability. The protomer for which W672 is not important is unique in the placement of its W666 sidechain between the other two protomers. We show that the unique orientation of this W666 sidechain azimuthally rotates its protomer away from the orientation it would have if the trimer were symmetric, resulting in the diminished interaction of this W672 with the rest of MPER₃. Our findings are consistent with our previous experimental study of W‐to‐A mutants of DAVEI. This suggests that DAVEI MPER may engage HIV‐1 Env to form a mixed trimer state in which one DAVEI MPER forms a trimer by displacing a more weakly interacting protomer of the endogenous Env MPER trimer.     

Complexes of HIV‐1 integrase with HAT proteins: Multiscale models,dynamics, and hypotheses on allosteric sites of inhibition

A new and very promising strategy for HIV drug discovery consists in blocking the multiple functional interactions between HIV‐1 integrase (IN) and its cellular cofactors. At present, this line of action is hindered by the absence of three‐dimensional structures of IN in complex with any of them. In this article, we developed a full‐length three‐dimensional structure of IN, including the highly flexible terminal residues 270–288, which are not experimentally solved. Additionally, we built models of IN complexed to the human acetyltransferases GCN5 and p300 based on available structural and mutagenesis data. Then, we studied the dynamical behavior of these models by means of the Coarse‐Grained Molecular Dynamics (CGMD) and Essential Dynamics (ED) to locate and characterize the nature of the largest collective motions. We found correlated motions involving distant regions of IN. Moreover, we found that these are influenced by the binding with the acetyltransferases (HATs). Taken together these findings suggest a way to affect the acetyltransferase binding by an allosteric type of inhibition and provide an important new approach for the drug design against HIV disease. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

Protein–protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein–protein–ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin‐dependent 3′,5′‐cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII–spectrin peptide (αII–spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C–mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM–protein complex under analysis. For the Ca²⁺–CaM, Ca²⁺–CaM–PDE1A, and Ca²⁺–CaM–MLCK complexes, CPZ apparent dissociation constants (K_ds) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC K_ds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII–spec) to Ca²⁺–hCaM M124C–mBBr quenched the fluorescence (K_d = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII–spec to a preformed Ca²⁺–hCaM M124C–mBBr–MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca²⁺–CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca²⁺–CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright © 2013 John Wiley & Sons, Ltd.     

The maturation of HIV‐1 protease precursor studied by discrete molecular dynamics

The equilibrium properties of a HIV‐1‐protease precursor are studied by means of an efficient molecular dynamics scheme, which allows for the simulation of the folding of the protein monomers and their dimerization into an active form and compare them with those of the mature protein. The results of the model provide, with atomic detail, an overall account of several experimental findings, including the NMR conformation of the mature dimer, the calorimetric properties of the system, the effects of the precursor tail on the dimerization constant, the secondary chemical shifts of the monomer, and the paramagnetic relaxation enhancement data associated with the conformations of the precursor. It is found that although the mature protein can dimerize in a unique, single way, the precursor populates several dimeric conformations in which monomers are always native‐like, but their binding can be non‐native. Proteins 2014; 82:633–639. © 2013 Wiley Periodicals, Inc.     

LuTHy: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ?L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.     

A method for reverse interactome analysis: High‐resolution mapping of interdomain interaction network in Dam1 complex and its specific disorganization based on the interaction domain expression

An experimental methodology that facilitates functional analysis of numerous protein–protein interactions, which have been found in genome‐wide interactome researches, has long been awaited. We propose herein an antagonistic inhibition‐based approach. The antagonizing polypeptide is generated in the course of interaction domain mapping based on yeast 2‐hybrid (Y2H) screening coupled with in vitro convergence of the Y2H‐selected fragments, which is performed in a formatted procedure. Using the coupled methodology, we first performed a high‐resolution mapping of an interdomain interaction network within budding yeast's Dam1 complex. Dam1 complex is a kinetochore protein complex composed of 10 essential subunits including Spc34p and Spc19p. The high‐resolution mapping revealed the overall network structure within the complex for the first time: Dam1 components form into two separated subnetworks on N‐terminal scaffolding domains of Spc34p and Spc19p, and the coiled‐coil interaction in their C‐terminal domains connects the subnetworks. Secondly, we show that the domain fragments converged in the high‐resolution mapping acted as potent inhibitors for the endogenous interactions when episomally overexpressed. The in vivo Dam1 interaction targeting with the fragments conferred a similar phenotype on the host cells; a critical and irreversible damage, which was accompanied with disturbed budding and chromosome mis‐segregation as a result of disorganized spindle. These phenotypes were strongly related to the cellular function of the Dam1 complex. The results and approach we demonstrated herein not only shed light on the Dam1 molecular architecture but also pave the road to reverse‐interactome analysis and discoveries of novel drugs that target disease‐related protein–protein interactions. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Molecular dynamics study of interaction and substrate channeling between neuron‐specific enolase and B‐type phosphoglycerate mutase

Phosphoglycerate mutase (PGM) and enolase are consecutive enzymes in the glycolytic pathway. We used molecular dynamics simulation to examine the interaction of human B‐type PGM (dPGM‐B) and neuron‐specific enolase (NSE). Specifically, we studied the interactions of 31 orientations of these enzymes by means of the effective energy function implicit solvation method. Interactions between active regions of the enzymes occurred preferentially, although the strongest interactions appeared to be between the back side of NSE and the active regions of dPGM‐B. Cleavage of 2PG from dPGM‐B was investigated, and the Ser¹⁴–Leu³⁰ loop of dPGM‐B is suggested as a cleavage site and, likely, another entrance site of a ligand. Substrate channeling between the enzymes was observed when NSE with its active regions Leu¹¹–Asn¹⁶, Arg⁴⁹–Lys⁵⁹, and Gly¹⁵⁵–Ala¹⁵⁸ covered the Ser¹⁴–Leu³⁰ loop of dPGM‐B. Analyses of the results make us believe that the channeling between PGM and enolase “benefits” from weak interaction. The probability of formation of channeling favorable complex is estimated to be up to 5%, while functional interaction between NSE and dPGM‐B might be as high as 20%. NSE and dPGM‐B functional interaction seems not to be isotype specific. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Experimental analysis of co‐evolution within protein complexes: The yeast exosome as a model

Extensive bioinformatics analysis suggests that the stability and function of protein complexes are maintained throughout evolution by coordinated changes (co‐evolution) of complex subunits. Yet, relatively little is known regarding the actual dynamics of such processes and the functional implications of co‐evolution within protein complexes, since most of the bioinformatics predictions were not analyzed experimentally. Here, we describe a systematic experimental approach that allows a step‐by‐step observation of the co‐evolution process in protein complexes. The exosome complex, an essential complex exhibiting a 3′→5′ RNA degradation activity, served as a model system. In this study, we show that exosome subunits diverged very early during fungal evolution. Interestingly, we found that despite significant differences in conservation between Rrp41 and Mtr3 both subunits exhibit similar divergence pattern and co‐evolutionary behavior through fungi evolution. Activity analysis of mutated exosomes exposes another layer of co‐evolution between the core subunits and RNA substrates. Overall, our approach allows the experimental analysis of co‐evolution within protein complexes and together with bioinformatics analysis can significantly deepen our understanding of the evolution of these complexes. Proteins 2013; 81:1997–2006. © 2013 Wiley Periodicals, Inc.