Ian Mitchelle S. de Vera

An alternate binding site for PPARγ ligands

PPARγ is a target for insulin sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes. Synthetic ligands have been designed to mimic endogenous ligand binding to a canonical ligand-binding pocket to hyperactivate PPARγ. Here we reveal that synthetic PPARγ ligands also bind to an alternate site, leading to unique receptor conformational changes that impact coregulator binding, transactivation and target gene expression. Using structure-function studies we show that alternate site binding occurs at pharmacologically relevant ligand concentrations, and is neither blocked by covalently bound synthetic antagonists nor by endogenous ligands indicating non-overlapping binding with the canonical pocket. Alternate site binding likely contributes to PPARγ hyperactivation in vivo, perhaps explaining why PPARγ full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPARγ activation by ligands and suggest that allosteric modulators could be designed to fine tune PPARγ activity without competing with endogenous ligands.

Conserved sequence-specific lincRNA–steroid receptor interactions drive transcriptional repression and direct cell fate

The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5 lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.

Archaeal JAB1/MPN/MOV34 metalloenzyme (HvJAMM1) cleaves ubiquitin‐like small archaeal modifier proteins (SAMPs) from protein‐conjugates

Proteins with JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) domains are widespread among all domains of life, yet poorly understood. Here we report the purification and characterization of an archaeal JAMM/MPN+ domain protein (HvJAMM1) from Haloferax volcanii that cleaves ubiquitin‐like small archaeal modifier proteins (SAMP1/2) from protein conjugates. HvJAMM1 cleaved SAMP1/2 conjugates generated in H. volcanii as well as isopeptide‐ and linear‐linked SAMP1–MoaE in purified form. Cleavage of linear linked SAMP1–MoaE was dependent on the presence of the SAMP domain and the C‐terminal VSGG motif of this domain. While HvJAMM1 was inhibited by size exclusion chromatography and metal chelators, its activity could be restored by addition of excess ZnCl2. HvJAMM1 residues (Glu31, His88, His90, Ser98 and Asp101) that were conserved with the JAMM/MPN+ active‐site motif were required for enzyme activity. Together, these results provide the first example of a JAMM/MPN+ zinc metalloprotease that independently catalyses the cleavage of ubiquitin‐like (isopeptide and linear) bonds from target proteins. In archaea, HvJAMM1 likely regulates sampylation and the pools of ‘free’ SAMP available for protein modification. HvJAMM1‐type proteins are thought to release the SAMPs from proteins modified post‐translationally as well as those synthesized as domain fusions.

Elucidating a Relationship between Conformational Sampling and Drug Resistance in HIV-1 Protease

Enzyme targets in rapidly replicating systems, such as retroviruses, commonly respond to drug-selective pressure with mutations arising in the active site pocket that limit inhibitor effectiveness by introducing steric hindrance or by eliminating essential molecular interactions. However, these primary mutations are disposed to compromising pathogenic fitness. Emerging secondary mutations, which are often found outside of the binding cavity, may or can restore fitness while maintaining drug resistance. The accumulated drug pressure selected mutations could have an indirect effect in the development of resistance, such as altering protein flexibility or the dynamics of protein-ligand interactions. Here, we show that accumulation of mutations in a drug-resistant HIV-1 protease (HIV-1 PR) variant, D30N/M36I/A71V, changes the fractional occupancy of the equilibrium conformational sampling ensemble. Correlations are made among populations of the conformational states, namely, closed-like, semiopen, and open-like, with inhibition constants, as well as kinetic parameters. Mutations that stabilize a closed-like conformation correlate with enzymes of lowered activity and with higher affinity for inhibitors, which is corroborated by a further increase in the fractional occupancy of the closed state upon addition of inhibitor or substrate-mimic. Cross-resistance is found to correlate with combinations of mutations that increase the population of the open-like conformations at the expense of the closed-like state while retaining native-like occupancy of the semiopen population. These correlations suggest that at least three states are required in the conformational sampling model to establish the emergence of drug resistance in HIV-1 PR. More importantly, these results shed light on a possible mechanism whereby mutations combine to impart drug resistance while maintaining catalytic activity.

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280 in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development.

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. (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra were acquired for each protease construct titrated with the same set of inhibitors. NMR (1)H-(15)N 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.

Pulsed EPR Distance Measurements in Soluble Proteins by Site‐Directed Spin Labeling (SDSL)

The resurgence of pulsed electron paramagnetic resonance (EPR) in structural biology centers on recent improvements in distance measurements using the double electron‐electron resonance (DEER) technique. This unit focuses on EPR‐based distance measurements by site‐directed spin labeling (SDSL) of engineered cysteine residues in soluble proteins, with HIV‐1 protease used as a model. To elucidate conformational changes in proteins, experimental protocols were optimized and existing data analysis programs were employed to derive distance‐distribution profiles. Experimental considerations, sample preparation, and error analysis for artifact suppression are also outlined herein. Curr. Protoc. Protein Sci. 74:17.17.1‐17.17.29.

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 IC(50) values are related to a parameter defined as |ΔC|, which is the relative change in the inhibitor-induced shift to the closed state. Specifically, a linear correlation is found between |ΔC| and the magnitude of the change in IC(50), 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.

Distal substitutions drive divergent DNA specificity among paralogous transcription factors through subdivision of conformational space

Significance Most organisms contain families of related proteins that evolved from duplication of an ancestral gene. Using the example of DNA binding by the steroid hormone receptors, this work examines the structural mechanisms by which these related proteins evolved separate functions during their history. We show that a functionally promiscuous ancestor was capable of accessing multiple protein conformations to bind disparate DNA sequences. This functional and conformational diversity were divided among daughter genes after gene duplication, allowing evolutionarily related proteins to generate disparate transcriptional outcomes in response to signaling input. Many genomes contain families of paralogs—proteins with divergent function that evolved from a common ancestral gene after a duplication event. To understand how paralogous transcription factors evolve divergent DNA specificities, we examined how the glucocorticoid receptor and its paralogs evolved to bind activating response elements [(+)GREs] and negative glucocorticoid response elements (nGREs). We show that binding to nGREs is a property of the glucocorticoid receptor (GR) DNA-binding domain (DBD) not shared by other members of the steroid receptor family. Using phylogenetic, structural, biochemical, and molecular dynamics techniques, we show that the ancestral DBD from which GR and its paralogs evolved was capable of binding both nGRE and (+)GRE sequences because of the ancestral DBD’s ability to assume multiple DNA-bound conformations. Subsequent amino acid substitutions in duplicated daughter genes selectively restricted protein conformational space, causing this dual DNA-binding specificity to be selectively enhanced in the GR lineage and lost in all others. Key substitutions that determined the receptors’ response element-binding specificity were far from the proteins’ DNA-binding interface and interacted epistatically to change the DBD’s function through DNA-induced allosteric mechanisms. These amino acid substitutions subdivided both the conformational and functional space of the ancestral DBD among the present-day receptors, allowing a paralogous family of transcription factors to control disparate transcriptional programs despite high sequence identity.

Effects of PRE and POST therapy drug-pressure selected mutations on HIV-1 protease conformational sampling.

Conformational sampling of pre‐ and post‐therapy subtype B HIV‐1 protease sequences derived from a pediatric subject infected via maternal transmission with HIV‐1 were characterized by double electron–electron resonance spectroscopy. The conformational ensemble of the PRE construct resembles native‐like inhibitor bound states. In contrast, the POST construct, which contains accumulated drug‐pressure selected mutations, has a predominantly semi‐open conformational ensemble, with increased populations of open‐like states. The single point mutant L63P, which is contained in PRE and POST, has decreased dynamics, particularly in the flap region, and also displays a closed‐like conformation of inhibitor‐bound states. These findings support our hypothesis that secondary mutations accumulate in HIV‐1 protease to shift conformational sampling to stabilize open‐like conformations, while maintaining the predominant semi‐open conformation for activity.