Correlating Conformational Shift Induction with Altered Inhibitor Potency in a Multidrug Resistant HIV‑1 Protease Variant

Inhibitor-induced conformational ensemble shifts in a multidrug resistant HIV-1 protease variant, MDR769, are characterized by site-directed spin labeling double electron–electron resonance spectroscopy. For MDR769 compared to the native enzyme, changes in inhibitor IC₅₀ values are related to a parameter defined as |Δ<i>C</i>|, which is the relative change in the inhibitor-induced shift to the closed state. Specifically, a linear correlation is found between |Δ<i>C</i>| and the magnitude of the change in IC₅₀, provided that inhibitor binding is not too weak. Moreover, inhibitors that exhibit MDR769 resistance no longer induce a strong shift to a closed conformational ensemble as seen previously in the native enzyme

Results from coevolution analysis of Hsp70 family members.

<p>On panel <b>A</b>, the heat map based on PSICOV covariance predictions is displayed. The white rectangular frame encloses the portion corresponding to interdomain co-variances. Residue pairs distinguished by strongest interdomain signals are listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-t001" target="_blank"><b>Table 1</b></a> and illustrated in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g007" target="_blank"><b>Figure 7</b></a>. Those residues exhibiting high cumulative interdomain coevolutionary propensities are labeled and displayed in space-filling representation (labeled on panel <b>B</b>) and listed in <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624.s007" target="_blank">Table S1</a></b>. The ribbon diagram is color-coded by the propensity of residues to exhibit coevolutionary patterns. NEF- and DnaJ-binding regions are highlighted. The DnaJ region is located mostly on the back of the area shown.</p

DnaK residues identified from GNM-mobility play a key role in interdomain allostery.

<p>(<b>A</b>) GNM-predicted mobility profile, <<i>M_i</i>>|<i>₁₀</i>, evaluated for the ATP-bound structure <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Kityk1" target="_blank">[25]</a> (<b>D</b> in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g001" target="_blank"><b>Figure 1</b></a>), residues 4-530. The boxes on the upper abscissa show the residue ranges of subdomains IA, IB, IIA and IIB of the NBD, and the SBD (same color-code as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g001" target="_blank"><b>Figure 1</b></a>). Minima (T417, D481 and G506 in the hinge region) on SBD and linker residues (V389-L392) are marked with red dots. (<b>B</b>) Color-coded ribbon diagram based on mobility (<i>red</i>: most mobile; <i>blue</i>: least mobile). The most mobile region on the ATPase domain (colored <i>pink-red</i>) is the NEF-binding subdomain IIB (residues G228-V309). The global mobility is based on the <i>m</i> = 10 GNM lowest frequency modes, which account for 40% of the overall dynamics. Three highly mobile C-terminal residues are truncated to permit a clearer visualization. (<b>C</b>) ATP-induced fluorescence shift of T417A DnaK variant relative to that of WT DnaK, shown as first derivatives of fluorescence spectra recorded in the absence (solid) and presence (dashed) of 1 mM ATP. Data for WT DnaK are shown in black and those for T417A DnaK in red. The ATP-induced blue shift of the T417A DnaK variant (3.3±0.5 nm) indicates that it adopts a domain-docked conformation in the presence of ATP to an extent comparable to WT DnaK (blue shift 3.5±0.5 nm). (<b>D</b>) Since cell growth at elevated temperatures strictly depends upon functional DnaK, the ability to grow after heat shock was used to assess the <i>in vivo</i> function of the T417A DnaK variant. Plates are shown that have been incubated at 43 °C after inoculation by serial dilutions of DnaK^–<i>E. coli</i> cells transformed either with an empty vector or with plasmids encoding the WT or T417A DnaK variant. Despite its ability to undergo the normal ATP-induced conformational rearrangement, T417A DnaK cannot support growth after heat shock. (<b>E</b>) The ATPase rates of the T417A DnaK variant relative to WT rates: basal (grey) and upon stimulation by a model peptides (red: p5, CALLLSAPRR, and blue: NR, NRLLLTG). Note that the peptide-induced interdomain allosteric communication responsible for the ATPase stimulation is significantly reduced in T417A DnaK. (<b>F</b>) Growth of <i>E. coli</i> cells that lack SecB is stringently dependent on functional DnaK <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Smock2" target="_blank">[20]</a>. Shown are relative cell densities of cells transformed with either the empty vector or plasmids encoding IPTG-inducible DnaK genes for WT or T417A DnaK variant, either in the presence of arabinose (grey), glucose (red), and glucose + IPTG (blue). Arabinose and glucose induce or repress (respectively) the expression of the chromosomal copies of the <i>dnaK</i> and <i>dnaJ</i> genes, which have been placed under the control of the P_BAD promoter, while IPTG induces expression of the plasmid-encoded DnaK variants. The optical density to which these cultures grow correlates with the degree of <i>in-vivo</i> functionality of the expressed DnaK variants.</p

Emerging network of interactions establishing the communication between the DnaJ binding site (near E217 and V389) and the ATP-binding site of DnaK.

<p>(<b>A</b>) Two interconnected pathways, also coupled to each other (via E171-D194 interaction) are shown, belonging to the respective subdomains IA (<i>red</i>) and IIA (<i>green</i>) of the DnaK NBD. (<b>B</b>) Most on-pathway residues are conserved. L177, which plays a central role is distinguished by its coevolution with V389 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g008" target="_blank"><b>Figure 8</b></a>) and high influence/sensitivity with respect to the majority of displayed residues (<b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624.s006" target="_blank">Figure S6</a></b>).</p

L177 mediates interdomain communication via a cascade of interactions between highly co-evolving residues, including V389 at interdomain linker and T417 and the global hinge.

<p>(<b>A</b>–<b>B</b>) Highly coevolving residues shown in <i>orange spheres</i>, except for linker residues V389 and L392, colored <i>red</i>. Coevolving pairs of amino acids and their relative spatial positions, shown by stick representation, bridging between the global hinge (T417) and ATP-binding site (via A17). Inter-residue distances are in Å. MIp matrix portions corresponding to these coevolving pairs are indicated. (<i>bottom</i>) (<b>C</b>) ATP-induced fluorescence shift of DnaK variant L177A relative to that of WT DnaK, as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g002" target="_blank">Figure 2c</a>. The ATP-induced blue shift of the L177A DnaK variant (2.0±0.5 nm) indicates that it is partially impaired in adopting a domain-docked conformation. (<b>D</b>) Growth-based functional assay at heat shock temperatures for L177A DnaK, as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g002" target="_blank">Figure 2d</a>. Consistent with the partial impairment of L177A DnaK in domain docking, this DnaK variant cannot support growth after heat shock. (<b>E</b>) ATPase rates of L177A DnaK variant relative to WT rates, as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g002" target="_blank"><b>Figure 2E</b></a>. Note that the peptide-induced interdomain allosteric communication responsible for the ATPase stimulation is significantly reduced in L177A DnaK. (<b>F</b>) Functional assay for L177A DnaK based on growth in the absence of SecB (as <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g002" target="_blank"><b>Figure 2F</b></a>), showing that this variant is severely impaired in <i>in-vivo</i> function relative to WT DnaK.</p

Close-up view of residue pairs distinguished by strong interdomain coevolutionary signals.

<p>Panels <b>A</b>–<b>C</b> display the structural position of residue pairs listed in the first part of <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-t001" target="_blank"><b>Table 1</b></a>, which make tertiary contacts. Panel <b>D</b> displays the location of all listed residues on the structure.</p

Hsp70 allosteric cycle.

<p>In the ADP-bound state (<b>B</b>, <i>bottom-left</i>), the SBD (<i>purple</i>) and NBD are loosely connected by a flexible interdomain linker. NBD subdomains are colored <i>red</i> (subdomain IA; residues 3-38; 112-184), <i>blue</i> (IB; residues 39-111), <i>green</i> (IIA; residues 185-228; 310-388) and <i>orange</i> (IIB; residues 229-309) as indicated in the middle diagram. Upon replacement of ADP by ATP (both in <i>yellow</i>, <i>space-filling</i>) and ensuing release of substrate (<i>cyan, space-filling</i>), an open-SBD conformer is assumed (<b>D</b>, <i>upper-right)</i>, where the α-helical lid is docked onto the NBD, exposing and opening the substrate-binding site. The two domains allosterically regulate each other through intermediate allosterically active states illustrated in <b>A</b> and <b>C </b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Zhuravleva1" target="_blank">[8]</a>: ATP binding decreases the substrate-binding affinity of the SBD; substrate-binding increases the ATPase activity of the NBD. The co-chaperones, DnaJ and GrpE, assist in the hydrolysis and nucleotide exchange steps, respectively. Diagrams <b>B</b> and <b>D</b> were generated using the PDB files 1DKX <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Zhu1" target="_blank">[7]</a> and 4B9Q <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Kityk1" target="_blank">[25]</a>, respectively. <b>A</b> and <b>C</b> were generated manually—as their complete structure is unknown—based on the two mentioned structures and also on 1DKG <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Harrison1" target="_blank">[65]</a> and 2KHO <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi.1003624-Bertelsen1" target="_blank">[16]</a>.</p

Sensitivity profile of global hinge site, and network of conserved interactions between effector residues at subdomain IA and the linker.

<p>(<b>A</b>) Sensitivity profile of D481 (representing the hinge region), showing the predominance of effector residues belonging to subdomain IA helix 6 (D148, Q152, K155) and central residues (G6, I18, V139, R167) in the neighboring four strands, and those on β SBD. (<b>B</b>) Location of these effectors on DnaK structure. (<b>C</b>) Network of interactions between effectors in the neighborhood of D481 (<i>yellow stick</i>). Some inter-residue distances are shown as dashed lines; units in Å. ATP is shown in <i>yellow stick</i>, in the back. (<b>D</b>) Sequence logo plot describing the conservation level of these effectors. Symbol sizes scale with the frequency of different amino acid types at the sequence position.</p

Summary of computational results and relevance to experimental observations.

<p><i>(*) See also the distant pairs that exhibit strong coevolution signals, listed in </i><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-t001" target="_blank"><i>Table 1</i></a><i>, and labeled in </i><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003624#pcbi-1003624-g007" target="_blank"><i>Fig 7D</i></a><i>.</i></p

Inhibitor-Induced Conformational Shifts and Ligand-Exchange Dynamics for HIV‑1 Protease Measured by Pulsed EPR and NMR Spectroscopy

Double electron–electron resonance (DEER) spectroscopy was utilized to investigate shifts in conformational sampling induced by nine FDA-approved protease inhibitors (PIs) and a nonhydrolyzable substrate mimic for human immunodeficiency virus type 1 protease (HIV-1 PR) subtype B, subtype C, and CRF_01 A/E. The ligand-bound subtype C protease has broader DEER distance profiles, but trends for inhibitor-induced conformational shifts are comparable to those previously reported for subtype B. Ritonavir, one of the strong-binding inhibitors for subtypes B and C, induces less of the closed conformation in CRF_01 A/E. ¹H–¹⁵N heteronuclear single-quantum coherence (HSQC) spectra were acquired for each protease construct titrated with the same set of inhibitors. NMR ¹H–¹⁵N HSQC titration data show that inhibitor residence time in the protein binding pocket, inferred from resonance exchange broadening, shifting or splitting correlates with the degree of ligand-induced flap closure measured by DEER spectroscopy. These parallel results show that the ligand-induced conformational shifts resulting from protein–ligand interactions characterized by DEER spectroscopy of HIV-1 PR obtained at the cryogenic temperature are consistent with more physiological solution protein–ligand interactions observed by solution NMR spectroscopy