7 research outputs found
Oxygen Pathways and Allostery in Monomeric Sarcosine Oxidase via Single-Sweep Free-Energy Reconstruction
Monomeric
sarcosine oxidase (MSOX) is a flavoprotein D-amino acid
oxidase with reported sarcosine and oxygen activation sites on the <i>re</i> and <i>si</i> faces of the flavin ring, respectively.
O<sub>2</sub> transport routes to the catalytic interior are not well
understood and are difficult to ascertain solely from MSOX crystal
structures. A composite free-energy method known as single-sweep is
used to map and thermodynamically characterize oxygen sites and routes
leading to the catalytically active Lys265 from the protein surface.
The result is a network of pathways and free energies within MSOX
illustrating that oxygen can access two free-energy minima on the <i>re</i> face of the reduced flavin from four separate solvent
portals. No such minimum is observed on the <i>si</i> face.
The pathways are geometrically similar for three major states of the
enzyme: (1) apo with a closed flavin cleft, (2) apo with an open flavin
cleft, and (3) inhibitor-bound with a closed flavin cleft. Interestingly,
free energies along these transport pathways display significantly
deeper minima when the substrate-mimicking inhibitor 2-furoic acid
is bound at the sarcosine site, even at locations far from this site.
This suggests a substrate-dependent allosteric modulation of the kinetics
of O<sub>2</sub> transport from the solvent to the active site
Dynamics of Lipids, Cholesterol, and Transmembrane 뱉Helices from Microsecond Molecular Dynamics Simulations
Extensive all-atom molecular dynamics
(âŒ24 ÎŒs total)
allowed exploration of configurational space and calculation of lateral
diffusion coefficients of the components of a protein-embedded, cholesterol-containing
model bilayer. The three model membranes are composed of an âŒ50/50
(by mole) dipalmitoylphosphatidylcholine (DPPC)/cholesterol bilayer
and contained an α-helical transmembrane protein (HIV-1 gp41
TM). Despite the high concentration of cholesterol, normal Brownian
motion was observed and the calculated diffusion coefficients (on
the order of 10<sup>â9</sup> cm<sup>2</sup>/s) are consistent
with experiments. Diffusion is sensitive to a variety of parameters,
and a temperature difference of âŒ4 K from thermostat artifacts
resulted in 2â10-fold differences in diffusion coefficients
and significant differences in lipid order, membrane thickness, and
unit cell area. Also, the specific peptide sequence likely underlies
the consistently observed faster diffusion in one leaflet. Although
the simulations here present molecular dynamics (MD) an order of magnitude
longer than those from previous studies, the three systems did not
approach ergodicity. The distributions of cholesterol and DPPC around
the peptides changed on the microsecond time scale, but not significantly
enough to thoroughly explore configurational space. These simulations
support conclusions of other recent microsecond MD in that even longer
time scales are needed for equilibration of model membranes and simulations
of more realistic cellular or viral bilayers
Full Kinetics of CO Entry, Internal Diffusion, and Exit in Myoglobin from Transition-Path Theory Simulations
We use Markovian milestoning molecular
dynamics (MD) simulations
on a tessellation of the collective variable space for CO localization
in myoglobin to estimate the kinetics of entry, exit, and internal
site-hopping. The tessellation is determined by analysis of the free-energy
surface in that space using transition-path theory (TPT), which provides
criteria for defining optimal milestones, allowing short, independent,
cell-constrained MD simulations to provide properly weighted kinetic
data. We coarse grain the resulting kinetic model at two levels: first,
using crystallographically relevant internal cavities and their predicted
interconnections and solvent portals; and second, as a three-state
side-path scheme inspired by similar models developed from geminate
recombination experiments. We show semiquantitative agreement with
experiment on entry and exit rates and in the identification of the
so-called âhistidine gateâ at position 64 through which
â90% of flux between solvent and the distal pocket passes.
We also show with six-dimensional calculations that the minimum free-energy
pathway of escape through the histidine gate is a âknock-onâ
mechanism in which motion of the ligand and the gate are sequential
and interdependent. In total, these results suggest that such TPT
simulations are indeed a promising approach to overcome the practical
time-scale limitations of MD to allow reliable estimation of transition
mechanisms and rates among metastable states
Computational Evaluation of HIVâ1 gp120 Conformations of Soluble Trimeric gp140 Structures as Targets for de Novo Docking of First- and Second-Generation Small-Molecule CD4 Mimics
Small-molecule CD4
mimics (SMCMâs) bind to the gp120 subunit
of the HIV-1 envelope glycoprotein (Env) and have been optimized to
block cell infection in vitro. The lack of the V1/2 and V3 loops and
the presence of the ÎČ2/3 and ÎČ20/21 strands (bridging
sheet) in the available structures of the monomeric gp120 core may
limit its applicability as a target for further synthetic optimization
of SMCM potency and/or breadth. Here, we employ a combination of binding-site
search, docking, estimation of proteinâligand interaction energy,
all-atom molecular dynamics, and ELISA-based CD4-binding competition
assays to create, characterize, and rationalize models of first- and
second-generation of SMCMâs bound to the distinct, trimeric
BG505 SOSIP.664 structures 4NCO and 4TVP containing V1/2 and V3 loops with no bridging sheet. We demonstrate
that the in silico neutralization of the highly conserved D368 is
necessary to obtain the correct orientation of SMCM in their binding
site when docking against the monomeric gp120 core. The computational
results correlate with IC<sub>50</sub>âs measured in CD4 binding
competition ELISA and with <i>K</i><sub>D</sub>âs
measured on gp120 core monomer. This supports the hypothesis that
the 4NCO trimeric
structure represents a viable target for further SMCMâs optimization
with advantages over both the 4TVP trimer and gp120 core monomer. Finally,
the docking protocol has been optimized to screen compounds that can
clearly interact with the highly conserved residue D368, increasing
the likelihood of future optimizations to arrive at SMCMâs
with a broader spectrum of activity
A Model of Peptide Triazole Entry Inhibitor Binding to HIVâ1 gp120 and the Mechanism of Bridging Sheet Disruption
Peptide
triazole (PT) entry inhibitors prevent HIV-1 infection by blocking
the binding of viral gp120 to both the HIV-1 receptor and the coreceptor
on target cells. Here, we used all-atom explicit solvent molecular
dynamics (MD) to propose a model for the encounter complex of the
peptide triazoles with gp120. Saturation transfer difference nuclear
magnetic resonance (STD NMR) and single-site mutagenesis experiments
were performed to test the simulation results. We found that docking
of the peptide to a conserved patch of residues lining the âF43
pocketâ of gp120 in a bridging sheet naiÌve gp120 conformation
of the glycoprotein led to a stable complex. This pose prevents formation
of the bridging sheet minidomain, which is required for receptorâcoreceptor
binding, providing a mechanistic basis for dual-site antagonism of
this class of inhibitors. Burial of the peptide triazole at the gp120
inner domainâouter domain interface significantly contributed
to complex stability and rationalizes the significant contribution
of hydrophobic triazole groups to peptide potency. Both the simulation
model and STD NMR experiments suggest that the I-X-W [where X is (2<i>S</i>,4<i>S</i>)-4-(4-phenyl-1<i>H</i>-1,2,3-triazol-1-yl)Âpyrrolidine]
tripartite hydrophobic motif in the peptide is the major contributor
of contacts at the gp120âPT interface. Because the model predicts
that the peptide Trp side chain hydrogen bonding with gp120 S375 contributes
to the stability of the PTâgp120 complex, we tested this prediction
through analysis of peptide binding to gp120 mutant S375A. The results
showed that a peptide triazole KR21 inhibits S375A with 20-fold less
potency than WT, consistent with predictions of the model. Overall,
the PTâgp120 model provides a starting point for both the rational
design of higher-affinity peptide triazoles and the development of
structure-minimized entry inhibitors that can trap gp120 into an inactive
conformation and prevent infection
Covalent Conjugation of a Peptide Triazole to HIVâ1 gp120 Enables Intramolecular Binding Site Occupancy
The HIV-1 gp120 glycoprotein is the
main viral surface protein
responsible for initiation of the entry process and, as such, can
be targeted for the development of entry inhibitors. We previously
identified a class of broadly active peptide triazole (PT) dual antagonists
that inhibit gp120 interactions at both its target receptor and coreceptor
binding sites, induce shedding of gp120 from virus particles prior
to hostâcell encounter, and consequently can prevent viral
entry and infection. However, our understanding of the conformational
alterations in gp120 by which PT elicits its dual receptor antagonism
and virus inactivation functions is limited. Here, we used a recently
developed computational model of the PTâgp120 complex as a
blueprint to design a covalently conjugated PTâgp120 recombinant
protein. Initially, a single-cysteine gp120 mutant, E275C<sub>YUâ2</sub>, was expressed and characterized. This variant retains excellent
binding affinity for peptide triazoles, for sCD4 and other CD4 binding
site (CD4bs) ligands, and for a CD4-induced (CD4i) ligand that binds
the coreceptor recognition site. In parallel, we synthesized a PEGylated
and biotinylated peptide triazole variant that retained gp120 binding
activity. An N-terminally maleimido variant of this PEGylated PT,
denoted AE21, was conjugated to E275C gp120 to produce the AE21âE275C
covalent conjugate. Surface plasmon resonance interaction analysis
revealed that the PTâgp120 conjugate exhibited suppressed binding
of sCD4 and 17b to gp120, signatures of a PT-bound state of envelope
protein. Similar to the noncovalent PTâgp120 complex, the covalent
conjugate was able to bind the conformationally dependent mAb 2G12.
The results argue that the PTâgp120 conjugate is structurally
organized, with an intramolecular interaction between the PT and gp120
domains, and that this structured state embodies a conformationally
entrapped gp120 with an altered bridging sheet but intact 2G12 epitope.
The similarities of the PTâgp120 conjugate to the noncovalent
PTâgp120 complex support the orientation of binding of PT to
gp120 predicted in the molecular dynamics simulation model of the
PTâgp120 noncovalent complex. The conformationally stabilized
covalent conjugate can be used to expand the structural definition
of the PT-induced âoffâ state of gp120, for example,
by high-resolution structural analysis. Such structures could provide
a guide for improving the subsequent structure-based design of inhibitors
with the peptide triazole mode of action
Structural and Functional Characterization of Indane-Core CD4-Mimetic Compounds Substituted with Heterocyclic Amines
The human immunodeficiency virus (HIV-1) envelope glycoprotein
(Env) trimer on the virion surface interacts with the host receptors,
CD4 and CCR5/CXCR4, to mediate virus entry into the target cell. CD4-mimetic
compounds (CD4mcs) bind the gp120 Env, block CD4 binding, and inactivate
Env. Previous studies suggested that a C(5)-methylamino methyl moiety
on a lead CD4mc, BNM-III-170, contributed to its antiviral potency.
By replacing the C(5) chain with differentially substituted pyrrolidine,
piperidine, and piperazine ring systems, guided by structural and
computational analyses, we found that the 5-position of BNM-III-170
is remarkably tolerant of a variety of ring sizes and substitutions,
both in regard to antiviral activity and sensitization to humoral
responses. Crystallographic analyses of representative analogues from
the pyrrolidine series revealed the potential for 5-substituents to
hydrogen bond with gp120 Env residue Thr 283. Further optimization
of these interactions holds promise for the development of CD4mcs
with greater potency