Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed ßα protein.

The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring ßα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable ßα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.

Probing the structural and dynamical effects of the charged residues of the TZF domain of TIS11d.

A member of the TTP family of proteins, TIS11d binds RNA with high specificity using a pair of CCCH-type tandem zinc fingers separated by a 18 residue long linker. Our previous work showed that the formation of hydrogen bonds between the C-terminal residue E220 and the residues of the linker region stabilized a compact structure of TIS11d in the absence of RNA. To investigate the role of the C-terminal residues in the structure of unbound TIS11d, the E220A mutant and the truncation mutant lacking the last two residues (D219/E220) were studied using molecular dynamics, NMR spectroscopy, and biochemical methods. This study confirmed the importance of the charged residues D219 and E220 in maintaining structural stability in unbound TIS11d and elucidated the underlying physical mechanisms. We observed a greater structural heterogeneity for the residues of the linker in the molecular dynamics trajectories of both mutant proteins relative to the wild-type. This heterogeneity was more pronounced in the D219/E220 deletion mutant than in the E220A mutant, indicating that a greater reduction of the charge of the C-terminus results in greater flexibility. In agreement with the increased flexibility and the reduced number of negatively charged residues of the D219/E220 deletion mutant, we measured more unfavorable entropic and a more favorable enthalpic contribution to the free energy of RNA binding in the mutant than in the wild-type protein. The relative orientation of the zinc fingers was stabilized by the electrostatic interaction between E220 and positively charged residues of the linker in TIS11d. In the E220A mutant, the relative orientation of the zinc fingers was less constrained, whereas in the D219/E220 deletion mutant, little orientational preference was observed. We posit that favorable electrostatic interactions provide a mechanism to promote preferential orientation of separate domains without imposing structural rigidity.

A conserved three-nucleotide core motif defines Musashi RNA binding specificity.

Musashi (MSI) family proteins control cell proliferation and differentiation in many biological systems. They are overexpressed in tumors of several origins, and their expression level correlates with poor prognosis. MSI proteins control gene expression by binding RNA and regulating its translation. They contain two RNA recognition motif (RRM) domains, which recognize a defined sequence element. The relative contribution of each nucleotide to the binding affinity and specificity is unknown. We analyzed the binding specificity of three MSI family RRM domains using a quantitative fluorescence anisotropy assay. We found that the core element driving recognition is the sequence UAG. Nucleotides outside of this motif have a limited contribution to binding free energy. For mouse MSI1, recognition is determined by the first of the two RRM domains. The second RRM adds affinity but does not contribute to binding specificity. In contrast, the recognition element for Drosophila MSI is more extensive than the mouse homolog, suggesting functional divergence. The short nature of the binding determinant suggests that protein-RNA affinity alone is insufficient to drive target selection by MSI family proteins.

The molecular basis of drug resistance against hepatitis C virus NS3/4A protease inhibitors.

Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors--telaprevir, danoprevir, vaniprevir and MK-5172--in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus.

A hydrophobic core stabilizes the residual structure in the RRM2 intermediate state of the ALS-linked protein TDP-43.

Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are prime targets for therapeutic interventions. In this study, we identified the core nucleus of structure for a folding intermediate in the second RNA recognition motif (RRM2) of the ALS-linked RNA-binding protein, TDP-43, using a combination of experimental and computational approaches. Urea equilibrium unfolding studies revealed that the RRM2 intermediate state consists of collapsed residual secondary structure localized to the N-terminal half of RRM2, while the C-terminus is largely disordered. Steered molecular dynamics simulations and mutagenesis studies yielded key stabilizing hydrophobic contacts that, when mutated to alanine, severely disrupt the overall fold of RRM2. In combination, these findings suggest a role for this RRM intermediate in normal TDP-43 function as well as serving as a template for misfolding and aggregation through the low stability and non-native secondary structure.

Molecular mechanisms of viral and host cell substrate recognition by hepatitis C virus NS3/4A protease.

Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.

Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody.

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.

An Engineered Antibody with Broad Protective Efficacy in Murine Models of SARS and COVID-19.

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. Here, we employed a directed evolution approach to engineer three SARS-CoV-2 antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains (RBDs) and neutralizes representative epidemic sarbecoviruses with remarkable potency. Structural and biochemical studies demonstrate that ADG-2 employs a unique angle of approach to recognize a highly conserved epitope overlapping the receptor binding site. In murine models of SARS-CoV and SARS-CoV-2 infection, passive transfer of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate for the treatment and prevention of SARS-CoV-2 and future emerging SARS-like CoVs.

Three Residues Make an Evolutionary Switch for Folding and RNA-Destabilizing Activity in the TTP Family of Proteins.

Tristetraprolin (TTP) binds to mRNA transcripts to promote their degradation. The TTP protein family in humans includes two other proteins, TIS11b and TIS11d. All three proteins contain a highly homologous RNA binding domain (RBD) that consists of two CCCH zinc fingers (ZFs). Both ZFs are folded in the absence of RNA in TIS11d and TIS11b. In TTP, however, only ZF1 adopts a stable fold. The focus of this study is to understand the origin and biological significance of the structural differences of the RBD. We identified three residues that affect the affinity for the structural Zn(2+) and determine the folding of ZF2 in the absence of RNA. We observed that the mRNA destabilizing activity of TTP was increased when the partially disordered RBD of TTP was replaced with the fully structured RBD of TIS11d, indicating that differences in the folded state of the RBD affect the activity of the proteins in the cell.

Structural Basis of the Disorder in the Tandem Zinc Finger Domain of the RNA-Binding Protein Tristetraprolin.

Tristetraprolin (TTP) and TIS11d are two human RNA-binding proteins that belong to the CCCH-type tandem zinc finger family. In the RNA-free state, TIS11d coordinates a zinc ion in each of its two fingers, while TTP coordinates a single zinc ion with the N-terminal zinc finger. We have previously identified three residues, located in the C-terminal half of a short α-helix in the second zinc finger, that control how structured the RNA-binding domain is in these two proteins: Y151, L152, and Q153 in TTP and H201, T202, and I203 in TIS11d. Here, we have used molecular dynamics, NMR spectroscopy, and other biochemical methods to investigate the role of these three residues in the stability of the RNA-binding domain. We found that the intrahelical hydrogen bond formed by the T202 hydroxyl group in the C-terminal zinc finger of TIS11d is necessary to allow for π-π stacking between the side chains of a conserved phenylalanine and the zinc-coordinating histidine. We demonstrated that the lack of this hydrogen bond in TTP is responsible for the reduced zinc affinity of the C-terminal zinc finger.

Allosteric inhibition of a stem cell RNA-binding protein by an intermediary metabolite.

Gene expression and metabolism are coupled at numerous levels. Cells must sense and respond to nutrients in their environment, and specialized cells must synthesize metabolic products required for their function. Pluripotent stem cells have the ability to differentiate into a wide variety of specialized cells. How metabolic state contributes to stem cell differentiation is not understood. In this study, we show that RNA-binding by the stem cell translation regulator Musashi-1 (MSI1) is allosterically inhibited by 18-22 carbon ω-9 monounsaturated fatty acids. The fatty acid binds to the N-terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. We identify stearoyl-CoA desaturase-1 as a MSI1 target, revealing a feedback loop between ω-9 fatty acid biosynthesis and MSI1 activity. We propose that other RRM proteins could act as metabolite sensors to couple gene expression changes to physiological state.