Rationalizing diverse binding mechanisms to the same protein fold: insights for ligand recognition and biosensor design.

The engineering of novel protein-ligand binding interactions, particularly for complex drug-like molecules, is an unsolved problem which could enable many practical applications of protein biosensors. In this work, we analyzed two engineer ed biosensors, derived from the plant hormone sensor PYR1, to recognize either the agrochemical mandipropamid or the synthetic cannabinoid WIN55,212-2. Using a combination of quantitative deep mutational scanning experiments and molecular dynamics simulations, we demonstrated that mutations at common positions can promote protein-ligand shape complementarity and revealed prominent differences in the electrostatic networks needed to complement diverse ligands. MD simulations indicate that both PYR1 protein-ligand complexes bind a single conformer of their target ligand that is close to the lowest free energy conformer. Computational design using a fixed conformer and rigid body orientation led to new WIN55,212-2 sensors with nanomolar limits of detection. This work reveals mechanisms by which the versatile PYR1 biosensor scaffold can bind diverse ligands. This work also provides computational methods to sample realistic ligand conformers and rigid body alignments that simplify the computational design of biosensors for novel ligands of interest.

Biophysical Rationale for the Selective Inhibition of PTP1B over TCPTP by Nonpolar Terpenoids.

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including cancer, autoimmunity, and neurological disorders. A high degree of structural similarity between their catalytic domains, however, has hindered the development of selective pharmacological agents. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over T-cell PTP (TCPTP), two PTPs with high sequence conservation. Here, we use molecular modeling, with supporting experimental validation, to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations suggest that PTP1B and TCPTP share a h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically essential WPD loop, which it links to the L-11 loop and neighboring α3 and α7 helices on the other side of the catalytic domain. Terpenoid binding to either of two proximal C-terminal sites─an α site and a ß site─can disrupt the allosteric network; however, binding to the α site forms a stable complex only in PTP1B. In TCPTP, two charged residues disfavor binding at the α site in favor of binding at the ß site, which is conserved between the two proteins. Our findings thus indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate more broadly how minor differences in the conservation of neighboring─yet functionally similar─allosteric sites can affect the selectivity of inhibitory scaffolds (e.g., fragments).

Analysis of neutral mutational drift in an allosteric enzyme.

Neutral mutational drift is an important source of biological diversity that remains underexploited in fundamental studies of protein biophysics. This study uses a synthetic transcriptional circuit to study neutral drift in protein tyrosine phosphatase 1B (PTP1B), a mammalian signaling enzyme for which conformational changes are rate limiting. Kinetic assays of purified mutants indicate that catalytic activity, rather than thermodynamic stability, guides enrichment under neutral drift, where neutral or mildly activating mutations can mitigate the effects of deleterious ones. In general, mutants show a moderate activity-stability tradeoff, an indication that minor improvements in the activity of PTP1B do not require concomitant losses in its stability. Multiplexed sequencing of large mutant pools suggests that substitutions at allosterically influential sites are purged under biological selection, which enriches for mutations located outside of the active site. Findings indicate that the positional dependence of neutral mutations within drifting populations can reveal the presence of allosteric networks and illustrate an approach for using synthetic transcriptional systems to explore these mutations in regulatory enzymes.

A biophysical rationale for the selective inhibition of PTP1B over TCPTP by nonpolar terpenoids.

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including type 2 diabetes, obesity, and cancer. However, a high degree of structural similarity between the catalytic domains of these enzymes has made the development of selective pharmacological inhibitors an enormous challenge. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over TCPTP, two PTPs with high sequence conservation. Here, we use molecular modeling with experimental validation to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations indicate that PTP1B and TCPTP contain a conserved h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically influential WPD loop, which it links to the L-11 loop and α 3 and α 7 helices-the C-terminal side of the catalytic domain. Terpenoid binding to either of two proximal allosteric sites-an α site and a ß site-can disrupt the allosteric network. Interestingly, binding to the α site forms a stable complex with only PTP1B; in TCPTP, where two charged residues disfavor binding at the α site, the terpenoids bind to the ß site, which is conserved between the two proteins. Our findings indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate, more broadly, how minor differences in the conservation of neighboring-yet functionally similar-allosteric sites can have very different implications for inhibitor selectivity.

Allosteric Inhibition of PTP1B by a Nonpolar Terpenoid.

Protein tyrosine phosphatases (PTPs) are promising drug targets for treating a wide range of diseases such as diabetes, cancer, and neurological disorders, but their conserved active sites have complicated the design of selective therapeutics. This study examines the allosteric inhibition of PTP1B by amorphadiene (AD), a terpenoid hydrocarbon that is an unusually selective inhibitor. Molecular dynamics (MD) simulations carried out in this study suggest that AD can stably sample multiple neighboring sites on the allosterically influential C-terminus of the catalytic domain. Binding to these sites requires a disordered α7 helix, which stabilizes the PTP1B-AD complex and may contribute to the selectivity of AD for PTP1B over TCPTP. Intriguingly, the binding mode of AD differs from that of the most well-studied allosteric inhibitor of PTP1B. Indeed, biophysical measurements and MD simulations indicate that the two molecules can bind simultaneously. Upon binding, both inhibitors destabilize the α7 helix by disrupting interactions at the α3-α7 interface and prevent the formation of hydrogen bonds that facilitate closure of the catalytically essential WPD loop. These findings indicate that AD is a promising scaffold for building allosteric inhibitors of PTP1B and illustrate, more broadly, how unfunctionalized terpenoids can engage in specific interactions with protein surfaces.