John M Louis

National Institutes of Health, 베서스다, Maryland, United States

Are you John M Louis?

Claim your profile

Publications (177)860.86 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: N-terminal self-cleavage (autoprocessing) of the HIV-1 protease precursor is crucial for liberating the active dimer. Under drug pressure, evolving mutations are predicted to modulate autoprocessing, and the reduced catalytic activity of the mature protease (PR) is likely compensated by enhanced conformational/dimer stability and reduced susceptibility to self-degradation (autoproteolysis). One such highly evolved, multi-drug resistant protease, PR20, bears 19 mutations contiguous to sites of autoproteolysis in retroviral proteases, namely clusters 1-3 comprising residues 30-37, 60-67 and 88-95, respectively, accounting for 11 of the 19 mutations. By systematically replacing corresponding clusters in PR with those of PR20, and vice versa, we assess their influence on the above properties and observe no strict correlation. A 10 to 35-fold decrease in cleavage efficiency of peptide substrates by PR20, relative to PR, is reflected by only ~4-fold decrease in the rate of Gag processing with no change in cleavage order. Importantly, optimal N-terminal autoprocessing requires all 19 PR20 mutations as evaluated in vitro using the model precursor TFR-PR20 in which PR is flanked by the transframe region. Substituting PR20 cluster 3 into TFR-PR (TFR-PR(PR20-3)) requires the presence of PR20 cluster 1 and/or 2 for autoprocessing. In accordance, substituting PR clusters 1 and 2 in TFR-PR20 affects the rate of autoprocessing more drastically (> 300-fold) compared to TFR-PR(PR20-3) because of the cumulative effect of eight non-cluster mutations present in TFR-PR20(PR-12). Overall, these studies imply that drug resistance involves a complex synchronized selection of mutations modulating all of the above properties governing PR regulation and function.
    Biochemistry 08/2015; DOI:10.1021/acs.biochem.5b00759 · 3.01 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Although Kramers' theory for diffusive barrier crossing on a 1D free energy profile plays a central role in landscape theory for complex biomolecular processes, it has not yet been rigorously tested by experiment. Here we test this 1D diffusion scenario with single molecule fluorescence measurements of DNA hairpin folding. We find an upper bound of 2.5 μs for the average transition path time, consistent with the predictions by theory with parameters determined from optical tweezer measurements.
    Physical Review Letters 07/2015; 115(1):018101. DOI:10.1103/PhysRevLett.115.018101 · 7.51 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: An extremely drug resistant mutant of HIV-1 protease (PR) bearing 20 mutations (PR20) has been studied with two potent antiviral investigational inhibitors. GRL-5010A and GRL-4410A were designed to introduce hydrogen bond interactions with the flexible flaps of the PR by incorporating gem-difluorines and alkoxy, respectively, at the C4 position of the bis-THF of darunavir. PR20 provides an excellent model for high level resistance, since clinical inhibitors are >1000-fold less active on PR20 than on wild-type enzyme. GRL-5010A and GRL-4410A show inhibition constants of 4.3  7.0 and 1.7  1.8 nM, respectively, for PR20, compared to the binding affinity of 41  1 nM measured for darunavir. Crystal structures of PR20 in complexes with the two inhibitors confirmed the new hydrogen bond interactions with Gly 48 in the flap of the enzyme. The two new compounds are more effective than darunavir in inhibiting mature PR20 and show promise for further development of antiviral agents targeting highly resistant PR mutants.
    Journal of Medicinal Chemistry 05/2015; 58(12). DOI:10.1021/acs.jmedchem.5b00474 · 5.48 Impact Factor
  • James L Baber · John M Louis · G Marius Clore
    [Show abstract] [Hide abstract]
    ABSTRACT: Pulsed double electron-electron resonance (DEER) provides pairwise P(r) distance distributions in doubly spin labeled proteins. We report that in protonated proteins, P(r) is dependent on the length of the second echo period T owing to local environmental effects on the spin-label phase memory relaxation time Tm . For the protein ABD, this effect results in a 1.4 Å increase in the P(r) maximum from T=6 to 20 μs. Protein A has a bimodal P(r) distribution, and the relative height of the shorter distance peak at T=10 μs, the shortest value required to obtain a reliable P(r), is reduced by 40 % relative to that found by extrapolation to T=0. Our results indicate that data at a series of T values are essential for quantitative interpretation of DEER to determine the extent of the T dependence and to extrapolate the results to T=0. Complete deuteration (99 %) of the protein was accompanied by a significant increase in Tm and effectively abolished the P(r) dependence on T. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    Angewandte Chemie International Edition in English 03/2015; 54(18). DOI:10.1002/anie.201500640 · 13.45 Impact Factor
  • James L. Baber · John M. Louis · G. Marius Clore
    [Show abstract] [Hide abstract]
    ABSTRACT: Pulsed double electron–electron resonance (DEER) provides pairwise P(r) distance distributions in doubly spin labeled proteins. We report that in protonated proteins, P(r) is dependent on the length of the second echo period T owing to local environmental effects on the spin-label phase memory relaxation time Tm. For the protein ABD, this effect results in a 1.4 Å increase in the P(r) maximum from T=6 to 20 μs. Protein A has a bimodal P(r) distribution, and the relative height of the shorter distance peak at T=10 μs, the shortest value required to obtain a reliable P(r), is reduced by 40 % relative to that found by extrapolation to T=0. Our results indicate that data at a series of T values are essential for quantitative interpretation of DEER to determine the extent of the T dependence and to extrapolate the results to T=0. Complete deuteration (99 %) of the protein was accompanied by a significant increase in Tm and effectively abolished the P(r) dependence on T.
    Angewandte Chemie 03/2015; 127(18). DOI:10.1002/ange.201500640
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ecto-domain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conforma-tion then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41's ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant inter-molecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of tri-merization of gp41's transmembrane helix to prevent complete dissociation of the trimer during the course of fusion.
    Journal of Biomolecular NMR 01/2015; · 3.31 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41's ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41's transmembrane helix to prevent complete dissociation of the trimer during the course of fusion.
    Journal of Biomolecular NMR 01/2015; 61(3-4). DOI:10.1007/s10858-015-9900-4 · 3.31 Impact Factor
  • Julien Roche · John M. Louis · Ad Bax
    Biophysical Journal 01/2015; 108(2):203a. DOI:10.1016/j.bpj.2014.11.1122 · 3.97 Impact Factor
  • Source
    Julien Roche · John M. Louis · Ad Bax
    [Show abstract] [Hide abstract]
    ABSTRACT: Flexibility of the glycine-rich flaps is known to be essential for catalytic activity of the HIV-1 protease, but their exact conformations at the different stages of the enzymatic pathway remain subject to much debate. Although hundreds of crystal structures of protease–inhibitor complexes have been solved, only about a dozen inhibitor-free protease structures have been reported. These latter structures reveal a large diversity of flap conformations, ranging from closed to semi-open to wide open. To evaluate the average structure in solution, we measured residual dipolar couplings (RDCs) and compared these to values calculated for crystal structures representative of the closed, semi-open, and wide-open states. The RDC data clearly indicate that the inhibitor-free protease, on average, adopts a closed conformation in solution that is very similar to the inhibitor-bound state. By contrast, a highly drug-resistant protease mutant, PR20, adopts the wide-open flap conformation.
    ChemBioChem 12/2014; 16(2). DOI:10.1002/cbic.201402585 · 3.06 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (>= 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.
    PLoS ONE 08/2014; 9(8):e104683. DOI:10.1371/journal.pone.0104683 · 3.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The molecular basis for high resistance to clinical inhibitors of HIV-1 protease (PR) was examined for the variant designated PRP51 that was selected for resistance to darunavir (DRV). High resolution crystal structures of PRP51 with the active site D25N mutation revealed a ligand free form and an inhibitor-bound form showing a unique binding site and orientation for DRV. This inactivating mutation is known to increase the dimer dissociation constant and DRV affinity of PR. The PRP51-D25N dimers were in the open conformation with widely separated flaps, as reported for other highly resistant variants. PRP51-D25N dimer bound two DRV molecules and showed larger separation of 8.7 Å between the closest atoms of the two flaps compared with 4.4 Å for the ligand-free structure of this mutant. The ligand-free structure, however, lacked van der Waals contacts between Ile50 and Pro81' from the other subunit in the dimer, unlike the majority of PR structures. DRV is bound inside the active site cavity, however, the inhibitor is oriented almost perpendicular to its typical position and exhibits only 2 direct hydrogen bond and two water-mediated interactions with atoms of PRP51-D25N compared with 11 hydrogen bond interactions seen for DRV bound in the typical position in wild-type enzyme. The atypical location of DRV may provide opportunities for design of novel inhibitors targeting the open conformation of PR drug resistant mutants.
    ACS Chemical Biology 04/2014; 9(6). DOI:10.1021/cb4008875 · 5.36 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. The actual fusion process involves a switch from a homotrimeric prehairpin intermediate conformation, consisting of parallel coiled-coil helices, to a postfusion state where the ectodomains are arranged as a trimer of helical hairpins, adopting a six-helix bundle (6HB) state. Here, we show by solution NMR spectroscopy that a water-soluble 6HB gp41 ectodomain binds to zwitterionic detergents that contain phosphocholine or phosphatidylcholine head groups and phospholipid vesicles that mimic T-cell membrane composition. Binding results in the dissociation of the 6HB and the formation of a monomeric state, where its two α-helices, N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), become embedded in the lipid-water interface of the virus and host cell. The atomic structure of the gp41 ectodomain monomer, based on NOE distance restraints and residual dipolar couplings, shows that the NHR and CHR helices remain mostly intact, but they completely lose interhelical contacts. The high affinity of the ectodomain helices for phospholipid surfaces suggests that unzippering of the prehairpin intermediate leads to a state where the NHR and CHR helices become embedded in the host cell and viral membranes, respectively, thereby providing a physical force for bringing these membranes into close juxtaposition before actual fusion.
    Proceedings of the National Academy of Sciences 02/2014; 111(9). DOI:10.1073/pnas.1401397111 · 9.81 Impact Factor
  • Source
    Julien Roche · John M. Louis · Ad Bax
    Biophysical Journal 01/2014; 106(2):708a. DOI:10.1016/j.bpj.2013.11.3929 · 3.97 Impact Factor
  • Source
    Biophysical Journal 01/2014; 106(2):707a. DOI:10.1016/j.bpj.2013.11.3925 · 3.97 Impact Factor
  • Source
    Biophysical Journal 01/2014; 106(2):439a. DOI:10.1016/j.bpj.2013.11.2475 · 3.97 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.
    PLoS ONE 11/2013; 8(11):e78187. DOI:10.1371/journal.pone.0078187 · 3.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. 1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. 2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. 3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20T26A and dimeric PR20 exhibit Tm values 6-7.5 °C higher than their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PRT26A by ~8 °C, similar to PR20T26A. However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PRD25N in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.
    Biochemistry 09/2013; 52(43). DOI:10.1021/bi400962r · 3.01 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Folding and unfolding rates for the ultrafast folding villin subdomain were determined from a photon-by-photon analysis of fluorescence trajectories in single molecule FRET experiments. One of the obstacles to measuring fast kinetics in single molecule fluorescence experiments is blinking of the fluorophores on a timescale that is not well separated from the process of interest. By incorporating acceptor blinking into a two-state kinetics model, we show that it is possible to extract accurate rate coefficients on the microsecond time scale for folding and unfolding using the maximum likelihood method of I.V. Gopich and A. Szabo. This method yields the most likely parameters of a given model that can reproduce the observed photon trajectories. The extracted parameters agree with both the decay rate of the donor-acceptor cross correlation function and the results of ensemble equilibrium and kinetic experiments using nanosecond laser temperature jump.
    Chemical Physics 08/2013; 422:229-237. DOI:10.1016/j.chemphys.2012.08.005 · 2.03 Impact Factor
  • Source
  • Source

Publication Stats

6k Citations
860.86 Total Impact Points

Institutions

  • 1988–2015
    • National Institutes of Health
      • • National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
      • • Laboratory of Chemical Physics (LCP)
      • • Laboratory of Experimental Gerontology (LEG)
      • • Laboratory of Cell and Developmental Biology
      베서스다, Maryland, United States
  • 1996–2014
    • The National Institute of Diabetes and Digestive and Kidney Diseases
      베서스다, Maryland, United States
    • Centre for Cellular and Molecular Biology
      Bhaganagar, Andhra Pradesh, India
  • 2009
    • Colorado State University
      • Biochemistry and Molecular Biology
      Fort Collins, CO, United States
  • 2007–2008
    • Georgia State University
      • • Department of Chemistry
      • • Department of Biology
      Atlanta, Georgia, United States
  • 1989–2004
    • National Cancer Institute (USA)
      Maryland, United States
  • 2003
    • National Eye Institute
      Maryland, United States
  • 1998
    • Case Western Reserve University
      • Department of Biochemistry
      Cleveland, Ohio, United States
  • 1992
    • NCI-Frederick
      Фредерик, Maryland, United States