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Eleven-nineteen leukemia protein (ENL) plays pivotal roles in the leukemogenesis. As a YEATS domain protein, ENL reads histone acylation marks and recruits key transcription factors to leukemic drivers such asHOXA9, MEIS1, andMYBand therefore promotes leukemia development. The histone-reading function of ENL has been proven essential in the onset and progression of several acute leukemias, suggesting a putative therapeutic window for ENL inhibition. In this study, we developed a phage-assisted, active site-directed ligand evolution (PADLE) approach for the identification of potent and selective ENL inhibitors, whereNε-butyryl-l-lysine (BuK) that possesses known target-protein interactions with the ENL YEATS domain was genetically incorporated into a phage display library to serve as a warhead to direct displayed peptides to the active site of ENL YEATS for enrichment. Using this novel strategy in combination with structure-activity relationship that replaced BuK with other ncAAs for de novo π-π-π stacking interactions with two aromatic residues in ENL YEATS, selective and potent ENL inhibitors with aKdvalue as low as 2.0 nM were identified. One pentapeptide inhibitortENL-S1fdisplayed selective inhibition of ENL over other YEATS domains as well as strong cellular target engagement and on-target effects in inhibiting leukemia cell growth and suppressing the expression of ENL target genes. As the first of its kind study, the current work opens a large avenue of research of using PADLE to develop selective and potent peptidyl inhibitors for a large variety of epigenetic reader proteins.

Phage-assisted, active site-directed ligand evolution (PADLE) is a recently developed technique that uses an amber codon-encoded noncanonical amino acid (ncAA) as an anchor to direct phage-displayed peptides to a target for an enhanced ligand identification process. 2-Amino-8-oxodecanoic acid (Aoda) is a ketone-containing ncAA residue in the macrocyclic peptide natural product apicidin that is a pan-inhibitor of Zn

The first step in transfer RNA (tRNA) maturation is the cleavage of the 5’ end of precursor transfer RNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein (RNP) complex with a catalytic RNA subunit or aprotein-onlyRNaseP(PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of protein-only RNase P that can be used as tools for studying the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP fromA. thalianaorganelles (PRORP1) using a high throughput fluorescence polarization (FP) cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 μM range were identified and analyzed. These compounds similarly inhibit human mtRNase P, a multi-subunit protein enzyme, and are 50-fold less potent against bacterial RNA-dependent RNase P. Biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor that targets the PRORP1-substrate complex while juglone acts as time-dependent inhibitor of PRORP1. X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. A model consistent with the kinetic data is that juglone binds to PRORP1 rapidly to form an inactive enzyme-inhibitor (EI) complex, and then undergoes a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships.

Di Si,1 Guochao Nie,2– 4 Tamiika K Hurst,1 Carol A Fierke,1 Raoul Kopelman1 1Department of Chemistry, University of Michigan, Ann Arbor, MI, USA; 2School of Physics and Telecommunication Engineering, Yulin Normal University, Yulin, People’s Republic of China; 3China-Ukraine Joint Research Center for Nano Carbon Black, Yulin, People’s Republic of China; 4Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology, Yulin, People’s Republic of ChinaCorrespondence: Guochao NieSchool of Physics and Telecommunication Engineering, Yulin Normal University, Yulin, People’s Republic of ChinaEmail bccu518@ylu.edu.cnRaoul KopelmanDepartment of Chemistry, University of Michigan, 930 N University Avenue, Ann Arbor, MI, USAEmail kopelman@umich.eduBackground: Due to its excellent biocompatibility, the polyacrylamide (PAAm) hydrogel has shown great potential for the immobilization of enzymes used in biomedical applications. The major challenge involved is to preserve, during the immobilization process, both the biological activity and the structural integrity of the enzymes. Here we report, for the first time, a proof-of-concept study for embedding active carbonic anhydrase (CA) into polyacrylamide (PAAm) nanogels. By immobilizing CA in these nanogels, we hope to provide important advantages, such as matrix protection of the CA as well as its targeted delivery, and also for potentially using these nanogels as zinc nano-biosensors, both in-vitro and in-vivo.Methods and Results: Two methods are reported here for CA immobilization: encapsulation and surface conjugation. In the encapsulation method, the common process was improved, so as to best preserve the CA, by 1) using a novel biofriendly nonionic surfactant system (Span 80/Tween 80/Brij 30) and 2) using an Al2O3 adsorptive filtration purification procedure. In the surface conjugation method, blank PAAm nanogels were activated by N-hydroxysuccinimide and the CA was cross-linked to the nanogels. The amount of active CA immobilized in the nanoparticles was quantified for both methods. Per 1 g nanogels, the CA encapsulated nanogels contain 11.3 mg active CA, while the CA conjugated nanogels contain 22.5 mg active CA. Also, the CA conjugated nanoparticles successfully measured free Zn2+ levels in solution, with the Zn2+ dissociation constant determined to be 9 pM.Conclusion: This work demonstrates universal methods for immobilizing highly fragile bio-macromolecules inside nanoparticle carriers, while preserving their structural integrity and biological activity. The advantages and limitations are discussed, as well as the potential biomedical applications.Keywords: PAAm hydrogel, nanogels, nanoparticles, carbonic anhydrase, encapsulation, conjugation, Zn2+

The phage-assisted, active site-directed ligand evolution (PADLE) is a recently developed technique that uses an amber codon-encoded noncanonical amino acid (ncAA) as an anchor to direct phage-displayed peptides to a target for an enhanced ligand identification process. 2-Amino-8-oxodecanoic acid (Aoda) is a ketone-containing ncAA residue in the macrocyclic peptide natural product apicidin that is a pan-inhibitor of Zn2+-dependent histone deacetylases (HDACs). Its ketone serves as an anchoring point to chelate the catalytic zinc ion in HDACs. Using a previously evolved Nε-acetyl-lysyl-tRNA synthetase in combination with tRNAPyl, we showed that Aoda was efficiently incorporated into proteins in Escherichia coli by amber suppression. By propagating an amber codon-obligate phagemid library in E. coli encoding Aoda, we generated an Aoda-containing phage-displayed peptide library. Using this library to conduct PADLE against HDAC8 revealed a 7-mer peptide GH8P01F1 with Aoda-flanking amino acid residues that match existing peptide sequences in identified HDAC8 substrates. Switching Aoda in GH8P01F1 to a more Zn2+-chelating ncAA S-2-amino-8-hydroxyamino-8-oxooctanoic acid (Asuha) led to an extremely potent GH8HA01 that has an HDAC8-inhibition Ki value as 0.67 nM. GH8HA01 and its 5-mer truncation analogue Ac-GH8HA01Δ1Δ7 that has an HDAC8-inhibition Ki value as 0.31 nM represent two most potent HDAC8 inhibitors that have been developed so far and both are highly selective against HDAC8 over the other three tested HDACs, demonstrating the great potential of using PADLE to identify highly potent and selective ligands for targets.

Numerous organic molecules are known to inhibit the main protease of SARS-CoV-2, (SC2Mpro), a key component in viral replication of the 2019 novel coronavirus. We explore the hypothesis that zinc ions, long used as a medicinal supplement and known to support immune function, bind to the SC2Mpro enzyme in combination with lipophilic tropolone and thiotropolone ligands, L, block substrate docking, and inhibit function. This study combines synthetic inorganic chemistry, in vitro protease activity assays, and computational modeling. While the ligands themselves have half maximal inhibition concentrations, IC50, for SC2Mpro in the 8-34 μM range, the IC50 values are ca. 100 nM for Zn(NO3)2 which are further enhanced in Zn-L combinations (59-97 nM). Isolation of the Zn(L)2 binary complexes and characterization of their ability to undergo ligand displacement is the basis for computational modeling of the chemical features of the enzyme inhibition. Blind docking onto the SC2Mpro enzyme surface using a modified Autodock4 protocol found preferential binding into the active site pocket. Such Zn-L combinations orient so as to permit dative bonding of Zn(L)+ to basic active site residues.

As the COVID-19 pathogen, SARS-CoV-2 relies on its main protease (MPro) for pathogenesis and replication. During the crystallographic analyses of MPro crystals that were exposed to the air, a uniquely Y-shaped, S-O-N-O-S-bridged posttranslational crosslink that connects three residues C22, C44, and K61 at their side chains was frequently observed. As a novel posttranslational modification, this crosslink serves as a redox switch to regulate the catalytic activity of MPro, a demonstrated drug target of COVID-19. The formation of this linkage leads to a much more opened active site that can be potentially targeted for the development of novel SARS-CoV-2 antivirals. The inactivation of MPro by this crosslink indicates that small molecules that lock MPro in the crosslinked form can be potentially used with other active site-targeting molecules such as paxlovid for synergistic effects in inhibiting the SARS-CoV-2 viral replication. Therefore, this new finding reveals a unique aspect of the SARS-CoV-2 pathogenesis and is potentially paradigm-shifting in our current understanding of the function of MPro and the development of its inhibitors as COVID-19 antivirals.

Mitochondrial diseases linked to mutations in mitochondrial (mt) tRNA sequences are common. However, the contributions of these tRNA mutations to the development of diseases is mostly unknown. Mutations may affect interactions with (mt)tRNA maturation enzymes or protein synthesis machinery leading to mitochondrial dysfunction. In human mitochondria, in most cases the first step of tRNA processing is the removal of the 5′ leader of precursor tRNAs (pre-tRNA) catalyzed by the three-component enzyme, mtRNase P. Additionally, one component of mtRNase P, mitochondrial RNase P protein 1 (MRPP1), catalyzes methylation of the R9 base in pre-tRNAs. Despite the central role of 5′ end processing in mitochondrial tRNA maturation, the link between mtRNase P and diseases is mostly unexplored. Here, we investigate how 11 different human disease-linked mutations in (mt)pre-tRNAIle, (mt)pre-tRNALeu(UUR), and (mt)pre-tRNAMet affect the activities of mtRNase P. We find that several mutations weaken the pre-tRNA binding affinity (KDs are approximately two- to sixfold higher than that of wild-type), while the majority of mutations decrease 5′ end processing and methylation activity catalyzed by mtRNase P (up to ∼55% and 90% reduction, respectively). Furthermore, all of the investigated mutations in (mt)pre-tRNALeu(UUR) alter the tRNA fold which contributes to the partial loss of function of mtRNase P. Overall, these results reveal an etiological link between early steps of (mt)tRNA-substrate processing and mitochondrial disease.

Significance Transcriptional activators represent a molecular recognition enigma. Their function in transcription initiation requires selective engagement of coactivators, yet the prevailing molecular recognition models propose this occurs via nonspecific intermolecular contacts. Here, mechanistic analysis of several related activator•coactivator complexes resolves this conundrum. In contrast to the expectations from nonspecific recognition models, even small sequence changes in the activators cause activator•coactivator complexes to undergo significant conformational redistribution, driven by specific intermolecular interactions and conformational changes in the coactivator itself. These unappreciated specific recognition mechanisms rationalize the high sequence variability of functional activators, opening new questions about the relationship between recognition and function., A key functional event in eukaryotic gene activation is the formation of dynamic protein–protein interaction networks between transcriptional activators and transcriptional coactivators. Seemingly incongruent with the tight regulation of transcription, many biochemical and biophysical studies suggest that activators use nonspecific hydrophobic and/or electrostatic interactions to bind to coactivators, with few if any specific contacts. Here a mechanistic dissection of a set of representative dynamic activator•coactivator complexes, comprised of the ETV/PEA3 family of activators and the coactivator Med25, reveals a different molecular recognition model. The data demonstrate that small sequence variations within an activator family significantly redistribute the conformational ensemble of the complex while not affecting overall affinity, and distal residues within the activator—not often considered as contributing to binding—play a key role in mediating conformational redistribution. The ETV/PEA3•Med25 ensembles are directed by specific contacts between the disordered activator and the Med25 interface, which is facilitated by structural shifts of the coactivator binding surface. Taken together, these data highlight the critical role coactivator plasticity plays in recognition of disordered activators and indicate that molecular recognition models of disordered proteins must consider the ability of the binding partners to mediate specificity.

Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5′-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNAPhe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNAPhe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.

Apolipoproteins are involved in pathological conditions of Alzheimer's disease (AD), and it has been reported that truncated apolipoprotein fragments and β-amyloid (Aβ) peptides coexist as neurotoxic heteromers within the plaques. Therefore, it is important to investigate these complexes at the molecular level to better understand their properties and roles in the pathology of AD. Here, we present a mechanistic insight into such heteromerization using a structurally homologue apolipoprotein fragment of apoA-I (4F) complexed with Aβ(M1-42) and characterize their toxicity. The 4F peptide slows down the aggregation kinetics of Aβ(M1-42) by constraining its structural plasticity. NMR and CD experiments identified 4F-Aβ(M1-42) heteromers comprised of unstructured Aβ(M1-42) and helical 4F. A uniform two-fold reduction in 15N/1H NMR signal intensities of Aβ(M1-42) with no observable chemical shift perturbation indicated the formation of a large complex, which was further confirmed by diffusion NMR experiments. Microsecond-scale atomistic molecular dynamics simulations showed that 4F interaction with Aβ(M1-42) is electrostatically driven and induces unfolding of Aβ(M1-42). Neurotoxicity profiling of Aβ(M1-42) complexed with 4F confirms a significant reduction in cell viability and neurite growth. Thus, the molecular architecture of heteromerization between 4F and Aβ(M1-42) discovered in this study provides evidence toward our understanding of the role of apolipoproteins or their truncated fragments in exacerbating AD pathology.

Histone deacetylases play important biological roles well beyond the deacetylation of histone tails. In particular, HDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as ɑ-tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of HDAC6 on a set of candidate unlabeled peptides. These served for the calibration of a structure-based substrate prediction protocol, Rosetta FlexPepBind, previously used for the successful substrate prediction of HDAC8 and other enzymes. A proteome-wide screen of reported acetylation sites using our calibrated protocol together with the enzymatic assay provide new peptide substrates and avenues to novel potential functional regulatory roles of this promiscuous, multi-faceted enzyme. In particular, we propose novel regulatory roles of HDAC6 in tumorigenesis and cancer cell survival via the regulation of EGFR/Akt pathway activation. The calibration process and comparison of the results between HDAC6 and HDAC8 highlight structural differences that explain the established promiscuity of HDAC6.

The first step in transfer RNA (tRNA) maturation is the cleavage of the 5' end of precursor tRNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of PRORP for use as tools to study the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 μM range were identified and analyzed. We found these compounds similarly inhibit human mtRNase P, a multisubunit protein enzyme and are 50-fold less potent against bacterial RNA-dependent RNase P. Our biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor targeting the PRORP1-substrate complex, while juglone acts as a time-dependent PRORP1 inhibitor. Additionally, X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. Finally, we propose a model consistent with the kinetic data that involves juglone binding to PRORP1 rapidly to form an inactive enzyme-inhibitor complex and then undergoing a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships.

Histone deacetylase (HDAC) enzymes that catalyze removal of acetyl-lysine post translational modifications are frequently post-translationally modified. HDAC8 is phosphorylated within the deacetylase domain at conserved residue serine 39 which leads to decreased catalytic activity. HDAC8 phosphorylation at S39 is unique in its location and function and may represent a novel mode of deacetylation regulation. To better understand the impact of phosphorylation of HDAC8 on enzyme structure and function, we performed crystallographic, kinetic, and molecular dynamics studies of the S39E HDAC8 phosphomimetic mutant. This mutation decreases deacetylation of peptides derived from acetylated nuclear and cytoplasmic proteins. However, the magnitude of the effect depends on the peptide sequence and the identity of the active site metal ion (Zn(II) vs Fe(II)) with the value of k(cat)/K(M) for the mutant decreasing 9- to >200-fold compared to wild-type HDAC8. Furthermore, the dissociation rate constant of the active site metal ion increases by ~15-fold. S39E HDAC8 was crystallized in complex with the inhibitor Droxinostat revealing that phosphorylation of S39, as mimicked by the glutamate side chain, perturbs local structure through distortion of the L1 loop. Molecular dynamics simulations of both S39E and phosphorylated S39 HDAC8 demonstrate that the perturbation of the L1 loop likely occurs because of the lost hydrogen bond between D29 and S39. Furthermore, the S39 perturbation causes structural changes that propagate through the protein scaffolding to influence function in the active site. These data demonstrate that phosphorylation plays an important regulatory role for HDAC8 by affecting ligand binding, catalytic efficiency and substrate selectivity.

In humans, β-amyloid and islet amyloid polypeptide (IAPP, also known as amylin) aggregations are linked to Alzheimer's disease and type-2 diabetes, respectively. There is significant interest in better understanding the aggregation process by using chemical tools. Here, we show the ability of a cationic polymethacrylate-copolymer (PMAQA) to quickly induce a β-hairpin structure and accelerate the formation of amorphous aggregates of β-amyloid-1-40, whereas it constrains the conformational plasticity of amylin for several days and slows down its aggregation at substoichiometric polymer concentrations. NMR experiments and microsecond scale atomistic molecular dynamics simulations reveal that PMAQA interacts with β-amyloid-1-40 residues spanning regions K16-V24 and A30-V40 followed by β-sheet induction. For amylin, it binds strongly close to the amyloid core domain (NFGAIL) and restrains its structural rearrangement. High-speed atomic force microscopy and transmission electron microscopy experiments show that PMAQA blocks the nucleation and fibrillation of amylin, whereas it induces the formation of amorphous aggregates of β-amyloid-1-40. Thus, the reported study provides a valuable approach to develop polymer-based amyloid inhibitors to suppress the formation of toxic intermediates of β-amyloid-1-40 and amylin.

Histone deacetylases play important biological roles well beyond the deacetylation of histone tails, and therefore have recently been renamed to acetyl-lysine deacetylases (KDACs). In particular, KDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as {square}-tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of KDAC6 on a set of candidate unlabeled peptides. These served for the calibration of a structure-based substrate prediction protocol, Rosetta FlexPepBind, previously used for the successful substrate prediction of KDAC8 and other enzymes. The calibration process and comparison of the results between KDAC6 and KDAC8 highlighted structural differences that explain the already reported promiscuity of KDAC6. A proteome-wide screen of reported acetylation sites using our calibrated protocol together with the enzymatic assay provide new peptide substrates and avenues to novel potential functional regulatory roles of this promiscuous, multi-faceted enzyme. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/431878v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@86bf82org.highwire.dtl.DTLVardef@87007corg.highwire.dtl.DTLVardef@1310594org.highwire.dtl.DTLVardef@1656965_HPS_FORMAT_FIGEXP M_FIG C_FIG

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M (Pro) ) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1M (Pro) ), we have designed and synthesized a series of SC2M (Pro) inhibitors that contain β-( S -2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2M (Pro) active site cysteine C145. All inhibitors display high potency with IC (50) values at or below 100 nM. The most potent compound MPI3 has as an IC (50) value as 8.5 nM. Crystallographic analyses of SC2M (Pro) bound to 7 inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549 cells. Two inhibitors MP5 and MPI8 completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 μM and A549 cells at 0.16-0.31 μM. Their virus inhibition potency is much higher than some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2M (Pro) inhibitors with extreme potency. Due to the urgent matter of the COVID-19 pandemic, MPI5 and MPI8 may be quickly advanced to preclinical and clinical tests for COVID-19.

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro), we have designed and synthesized a series of SC2MPro inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active site cysteine C145. All inhibitors display high potency with IC50 values at or below 100 nM. The most potent compound MPI3 has as an IC50 value as 8.5 nM. Crystallographic analyses of SC2MPro bound to 7 inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549 cells. Two inhibitors MP5 and MPI8 completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 μM and A549 cells at 0.16-0.31 μM. Their virus inhibition potency is much higher than some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with extreme potency. Due to the urgent matter of the COVID-19 pandemic, MPI5 and MPI8 may be quickly advanced to preclinical and clinical tests for COVID-19.

A key functional event in eukaryotic gene activation is the formation of dynamic protein-protein interaction networks between transcriptional activators and transcriptional coactivators. Seemingly incongruent with the tight regulation of transcription, many biochemical and biophysical studies suggest that activators use nonspecific hydrophobic and/or electrostatic interactions to bind to coactivators, with few if any specific contacts. Here a mechanistic dissection of a set of representative dynamic activator•coactivator complexes, comprised of the ETV/PEA3 family of activators and the coactivator Med25, reveals a different molecular recognition model. The data demonstrate that small sequence variations within an activator family significantly redistribute the conformational ensemble of the complex while not affecting overall affinity, and distal residues within the activator—not often considered as contributing to binding—play a key role in mediating conformational redistribution. The ETV/PEA3•Med25 ensembles are directed by specific contacts between the disordered activator and the Med25 interface, which is facilitated by structural shifts of the coactivator binding surface. Taken together, these data highlight the critical role coactivator plasticity plays in recognition of disordered activators, and indicates that molecular recognition models of disordered proteins must consider the ability of the binding partners to mediate specificity.

Significance Transcriptional coactivators and their partner transcription factors have been labeled as intrinsically disordered, fuzzy, and undruggable. We propose that the identification of conserved mechanisms of engagement between coactivators and their cognate activators should provide general principles for small-molecule modulator discovery. Here, we show that the structurally divergent coactivator Med25 forms short-lived and dynamic complexes with three different transcriptional activators and that conformational shifts are mediated by a flexible substructure of two dynamical helices and flanking loops. Analogous substructures are found across coactivators. Further, targeting one of the flexible structures with a small molecule modulates Med25–activator complexes. Thus, the two conclusions of the work are actionable for the discovery of small-molecule modulators of this functionally important protein class., Transcriptional coactivators are a molecular recognition marvel because a single domain within these proteins, the activator binding domain or ABD, interacts with multiple compositionally diverse transcriptional activators. Also remarkable is the structural diversity among ABDs, which range from conformationally dynamic helical motifs to those with a stable core such as a β-barrel. A significant objective is to define conserved properties of ABDs that allow them to interact with disparate activator sequences. The ABD of the coactivator Med25 (activator interaction domain or AcID) is unique in that it contains secondary structural elements that are on both ends of the spectrum: helices and loops that display significant conformational mobility and a seven-stranded β-barrel core that is structurally rigid. Using biophysical approaches, we build a mechanistic model of how AcID forms binary and ternary complexes with three distinct activators; despite its static core, Med25 forms short-lived, conformationally mobile, and structurally distinct complexes with each of the cognate partners. Further, ternary complex formation is facilitated by allosteric communication between binding surfaces on opposing faces of the β-barrel. The model emerging suggests that the conformational shifts and cooperative binding is mediated by a flexible substructure comprised of two dynamic helices and flanking loops, indicating a conserved mechanistic model of activator engagement across ABDs. Targeting a region of this substructure with a small-molecule covalent cochaperone modulates ternary complex formation. Our data support a general strategy for the identification of allosteric small-molecule modulators of ABDs, which are key targets for mechanistic studies as well as therapeutic applications.

Histone deacetylases (HDACs) catalyze deacetylation of acetyl-lysine residues within proteins. To date, HDAC substrate specificity and selectivity have been largely estimated using peptide substrates. However, it is unclear whether peptide substrates accurately reflect the substrate selectivity of HDAC8 toward full-length proteins. Here, we compare HDAC8 substrate selectivity in the context of peptides, full-length proteins, and protein-nucleic acid complexes. We demonstrate that HDAC8 catalyzes deacetylation of tetrameric histone (H3/H4) substrates with catalytic efficiencies that are 40-300-fold higher than those for corresponding peptide substrates. Thus, we conclude that additional contacts with protein substrates enhance catalytic efficiency. However, the catalytic efficiency decreases for larger multiprotein complexes. These differences in HDAC8 substrate selectivity for peptides and full-length proteins suggest that HDAC8 substrate preference is based on a combination of short- and long-range interactions. In summary, this work presents detailed kinetics for HDAC8-catalyzed deacetylation of singly-acetylated, full-length protein substrates, revealing that HDAC8 substrate selectivity is determined by multiple factors. These insights provide a foundation for understanding recognition of full-length proteins by HDACs.

Identification of the function of metal ions and the RNA moieties, particularly nucleobases, that bind metal ions are important questions in RNA catalysis. Here we combine single-atom and abasic substitutions to probe functions of conserved nucleobases in ribonuclease P (RNase P). Structural and biophysical studies of bacterial RNase P propose direct coordination of metal ions by the nucleobases of conserved uridine and guanosine in helix P4 of the RNA subunit (P RNA). To biochemically probe the function of metal ion interactions, we substituted the universally conserved bulged uridine (U51) in the P4 helix of circularly permuted Bacillus subtilis P RNA with 4-thiouridine, 4-deoxyuridine and abasic modifications and G378/379 with 2-aminopurine, N7-deazaguanosine, and 6-thioguanosine. The functional group modifications of U51 decrease RNase P-catalyzed phosphodiester bond cleavage 16- to 23-fold, as measured by the single-turnover cleavage rate constant. The activity of the 4-thiouridine RNase P is partially rescued by addition of Cd(II) or Mn(II) ions. This is the first time a metal-rescue experiment provides evidence for inner-sphere divalent metal ion coordination with a nucleobase. Modifications of G379 modestly decrease the cleavage activity of RNase P, suggesting outer-sphere coordination of O6 on G379 to a metal ion. These data provide biochemical evidence for catalytically important interactions of the P4 helix of P RNA with metal ions, demonstrating that the bulged uridine coordinates at least one catalytic metal ion through an inner-sphere interaction. The combination of single-atom and abasic nucleotide substitutions provides a powerful strategy to probe functions of conserved nucleobases in large RNAs.

The histone deacetylase family comprises 18 enzymes that catalyze deacetylation of acetylated lysine residues, however, the specificity and substrate profile of each enzyme remains largely unknown. Due to transient enzyme-substrate interactions, conventional co-immunoprecipitation methods frequently fail to identify enzyme-specific substrates. Additionally, compensatory mechanisms often limit the ability of knockdown or chemical inhibition studies to achieve significant fold-changes observed by acetylation proteomics methods. Furthermore, measured alterations do not guarantee a direct link between enzyme and substrate. Here we present a chemical crosslinking strategy that incorporates a photo-reactive, non-natural amino acid, p-benzoyl-L-phenylalanine, into various positions of the structurally characterized isozyme histone deacetylase 8 (HDAC8). After covalent capture, co-immunoprecipitation, and mass spectrometric analysis, we identified a subset of HDAC8 substrates from human cell lysates, which were further validated for catalytic turnover. Overall, this chemical-crosslinking approach identified novel HDAC8 specific substrates with greater catalytic efficiency, thus presenting a general strategy for unbiased deacetylase substrate discovery.

Protein-only ribonuclease P (PRORP) is an enzyme responsible for catalyzing the 5′ end maturation of precursor transfer ribonucleic acids (pre-tRNAs) encoded by various cellular compartments in many eukaryotes. PRORPs from plants act as single-subunit enzymes and have been used as a model system for analyzing the function of the metazoan PRORP nuclease subunit, which requires two additional proteins for efficient catalysis. There are currently few molecular details known about the PRORP–pre-tRNA complex. Here, we characterize the determinants of substrate recognition by the single subunit Arabidopsis thaliana PRORP1 and PRORP2 using kinetic and thermodynamic experiments. The salt dependence of binding affinity suggests 4–5 contacts with backbone phosphodiester bonds on substrates, including a single phosphodiester contact with the pre-tRNA 5′ leader, consistent with prior reports of short leader requirements. PRORPs contain an N-terminal pentatricopeptide repeat (PPR) domain, truncation of which results in a >30-fold decrease in substrate affinity. While most PPR-containing proteins have been implicated in single-stranded sequence-specific RNA recognition, we find that the PPR motifs of PRORPs recognize pre-tRNA substrates differently. Notably, the PPR domain residues most important for substrate binding in PRORPs do not correspond to positions involved in base recognition in other PPR proteins. Several of these residues are highly conserved in PRORPs from algae, plants, and metazoans, suggesting a conserved strategy for substrate recognition by the PRORP PPR domain. Furthermore, there is no evidence for sequence-specific interactions. This work clarifies molecular determinants of PRORP–substrate recognition and provides a new predictive model for the PRORP–substrate complex.

Histone deacetylase 8, part of a broad class of proteins responsible for regulating transcription and many other cellular processes and directly linked to a host of human disease through its mis-function, has been canonically described as a zinc-based mettalo-enzyme for many years. Recent evidence, however, has linked this protein to iron incorporation, loaded through transient interactions with the poly r(C)-binding protein 1, a metallo-chaperone and storage protein. In this report, we construct and deploy an electrospray-mass spectrometry based assay aimed at quantifying the interaction strength between these two weakly-associated proteins, as well as the zinc and iron associated form of the histone deacetylase. Despite challenges derived from artifact protein complexes derived from the electrospray process, we use carefully-constructed positive and negative control experiments, along with detailed measurements of protein ionization efficiency to validate our dissociation constant measurements for protein dimers in this size range. Furthermore, our data strongly support that complexes between histone deacetylase 8 and poly r(C)-binding protein 1 are specific, and that they are equally strong when both zinc and iron-loaded proteins are involved, or perhaps mildly promoted in the latter case, suggesting an in vivo role for the non-canonical, iron-incorporated histone deacetylase.

Pancreatic ductal adenocarcinoma is an aggressive cancer with limited treatment options(1). Approximately 10% of cases exhibit familial predisposition, but causative genes are not known in most families(2). We perform whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma and identify a germline truncating mutation in the member of the RAS oncogene family-like (3) (RABL3) gene. Heterozygous rabl3 mutant zebrafish show increased susceptibility to cancer formation. Transcriptomic and mass spectrometry approaches implicate RABL3 in RAS pathway regulation and identify an interaction with RAP1GDS1 (SmgGDS), a chaperone regulating prenylation of RAS GTPases(3). Indeed, the truncated mutant RABL3 protein accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Finally, evidence in patient cohorts with developmental disorders implicates germline RABL3 mutations in RASopathy syndromes. Our studies identify RABL3 mutations as a target for genetic testing in cancer families and uncover a mechanism for dysregulated RAS activity in development and cancer.

Apolipoproteins are involved in pathological conditions of Alzheimer’s disease (AD), truncated apolipoprotein fragments and β-amyloid (Aβ) peptides coexist as neurotoxic heteromers within the plaques. Therefore, it is important to investigate these complexes at the molecular level to better understand their properties and roles in the pathology of AD. Here, we present a mechanistic insight into such heteromerization using a structurally homologue apolipoprotein fragment of apoA-I (4F) complexed with Aβ(M1-42) and characterize their toxicity. The 4F peptide slows down the aggregation kinetics of Aβ(M1-42) by constraining its structural plasticity. NMR and CD experiments identified 4F-Aβ(M1-42) heteromers as being comprised of unstructured Aβ(M1-42) and helical 4F. A uniform ≈2-fold reduction in Aβ4215N/1H NMR signal intensities with no observable chemical shift perturbation indicated the formation of a large complex, which was further confirmed by diffusion NMR experiments. Microsecond scale atomistic molecular dynamics simulations showed that 4F interaction with Aβ(M1-42) is electrostatically driven and induces unfolding of Aβ(M1-42). Neurotoxicity profiling of Aβ(M1-42) complexed with 4F confirms a significant reduction in cell-viability and neurite growth. The molecular architecture of heteromerization between 4F and Aβ(M1-42) discovered in this study provides evidence towards our understanding of the role of apolipoproteins or their truncated fragments in exacerbating AD pathology.

Ras family small GTPases undergo prenylation (such as farnesylation) for proper localization to the plasma membrane, where they can initiate oncogenic signaling pathways. Small GTP-binding protein GDP-dissociation stimulator (SmgGDS) proteins are chaperones that bind and traffic small GTPases, although their exact cellular function is unknown. Initially, SmgGDS proteins were classified as guanine nucleotide exchange factors, but recent findings suggest that SmgGDS proteins also regulate prenylation of small GTPases in vivo in a substrate-selective manner. SmgGDS-607 recognizes the polybasic region and the CAAX box of several small GTPases and inhibits prenylation by impeding their entry into the geranylgeranylation pathway. Here, using recombinant and purified enzymes for prenylation and protein-binding assays, we demonstrate that SmgGDS-607 differentially regulates farnesylation of several small GTPases. SmgGDS-607 inhibited farnesylation of some proteins, such as DiRas1, by sequestering the protein and limiting modification catalyzed by protein farnesyltransferase (FTase). We found that the competitive binding affinities of the small GTPase for SmgGDS-607 and FTase dictate the extent of this inhibition. Additionally, we discovered that SmgGDS-607 increases the rate of farnesylation of HRas by enhancing product release from FTase. Our work indicates that SmgGDS-607 binds to a broad range of small GTPases and does not require a PBR for recognition. Together, these results provide mechanistic insight into SmgGDS-607–mediated regulation of farnesylation of small GTPases and suggest that SmgGDS-607 has multiple modes of substrate recognition.

A first step in processing mitochondrial precursor tRNA (pre-tRNA) is cleavage of the 5’ leader catalyzed by ribonuclease P (RNase P). Human mitochondrial RNase P (mtRNase P) is composed of three protein subunits: mitochondrial RNase P protein (MRPP) 1, 2 and 3. Even though MRPP3 is the metallonuclease subunit responsible for catalysis, cleavage is observed only in the presence of the MRPP1/2 subcomplex. To understand the functional role of MRPP1/2, we reconstituted human mitochondrial RNase P in vitro and performed kinetic and thermodynamic analyses. MRPP1/2 significantly enhances both the catalytic activity and the apparent substrate affinity of mtRNase P. Additionally, pull-down and binding data demonstrate synergy between binding pre-tRNA and formation of a catalytically active MRPP1/2/3 complex. These data suggest that conformational changes in the MRPP1/2-pre-tRNA complex lead to protein-protein or protein-RNA interactions that increase both MRPP3 recognition and cleavage efficiency. This work presents the first kinetic model for human mtRNase P, providing a fundamental framework for the function of MRPP1/2 for recognition and processing of pre-tRNA.

Ribonuclease P (RNase P) catalyzes the cleavage of leader sequences from precursor tRNA (pre-tRNA). Typically, these enzymes are ribonucleic protein complexes that are found in all domains of life. However, a new class of RNase P has been discovered that is composed entirely of protein, termed protein-only RNase P (PRORP). To investigate the molecular determinants of PRORP substrate recognition, we measured the binding affinities and cleavage kinetics of Arabidopsis PRORP1 for varied pre-tRNA substrates. This analysis revealed that PRORP1 does not make significant contacts within the trailer or beyond N−1 of the leader, indicating that this enzyme recognizes primarily the tRNA body. To determine the extent to which sequence variation within the tRNA body modulates substrate selectivity and to provide insight into the evolution and function of PRORP enzymes, we measured the reactivity of the three Arabidopsis PRORP isozymes (PRORP1–3) with four pre-tRNA substrates. A 13-fold range in catalytic efficiencies (104–105 M−1 s−1) was observed, demonstrating moderate selectivity for pre-tRNA substrates. Although PRORPs bind the different pre-tRNA species with affinities varying by as much as 100-fold, the three isozymes have similar affinities for a given pre-tRNA, suggesting similar binding modes. However, PRORP isozymes have varying degrees of cleavage fidelity, which is dependent on the pre-tRNA species and the presence of a 3′-discriminator base. This work defines molecular determinants of PRORP substrate recognition that provides insight into this new class of RNA processing enzymes.

HDAC8 is a member of the family of histone deacetylases (HDACs) that catalyze the deacetylation of acetyl lysine residues within histone and non-histone proteins. The recent identification of novel non-histone HDAC8 substrates such as SMC3, ERRα, and ARID1A indicates a complex functionality of this enzyme in cellular homeostasis. To discover additional HDAC8 substrates, we developed a comprehensive, structure-based approach based on Rosetta FlexPepBind, a protocol that evaluates peptide-binding ability to a receptor from structural models of this interaction. Here we adapt this protocol to identify HDAC8 substrates using peptide sequences extracted from proteins with known acetylated sites. The many new in vitro HDAC8 peptide substrates identified in this study suggest that numerous cellular proteins are HDAC8 substrates, thus expanding our view of the acetylome and its regulation by HDAC8.

Epigenetic regulation is a key factor in cellular homeostasis. Post-translational modifications (PTMs) are a central focus of this regulation as they function as signaling markers within the cell. Lysine acetylation is a dynamic, reversible PTM that has garnered recent attention due to alterations in various types of cancer. Acetylation levels are regulated by two opposing enzyme families: lysine acetyltransferases (KATs) and histone deacetylases (HDACs). HDACs are key players in epigenetic regulation and have a role in the silencing of tumor suppressor genes. The dynamic equilibrium of acetylation makes HDACs attractive targets for drug therapy. However, substrate selectivity and biological function of HDAC isozymes is poorly understood. This review outlines the current understanding of the roles and specific epigenetic interactions of the metal-dependent HDACs in addition to their roles in cancer.

Attempts to identify the prenyl-proteome of cells or changes in prenylation following drug treatment have used “clickable” alkyne-modified analogs of the lipid substrates farnesyl- and geranylgeranyl-diphosphate (FPP and GGPP). We characterized the reactivity of four alkyne-containing analogs of FPP with purified protein farnesyltransferase and a small library of dansylated peptides using an in vitro continuous spectrofluorimetric assay. These analogs alter prenylation specificity and reactivity suggesting that in vivo results obtained using these FPP analogs should be interpreted cautiously.

The deposition of aggregates of human islet amyloid polypeptide (hIAPP) has been correlated with the death of β-cells in type II diabetes mellitus. The actual molecular mechanism of cell death remains largely unknown; however, it has been postulated that the process of aggregation from monomeric hIAPP is closely involved. A possible cause of cellular toxicity may be through the disruption of structural integrity of the cell membrane by IAPP. Herein, a water-soluble curcumin derivative, CurDAc, is used to investigate the mitigation of hIAPP aggregation in the absence and presence of lipid membrane.

Polymethacrylate-copolymer (PMA) encased lipid-nanodiscs (∼10 nm) and macro-nanodiscs (>15 nm) are used to study Aβ1–40 aggregation. We demonstrate that PMA-nanodiscs form a ternary association with Aβ and regulate its aggregation kinetics by trapping intermediates. Results demonstrating the reduced neurotoxicity of nanodisc-bound Aβ oligomers are also reported.

Human mitochondrial ribonuclease P (mtRNase P) is an essential three protein complex that catalyzes the 5’ end maturation of mitochondrial precursor tRNAs (pre-tRNAs). MRPP3 (Mitochondrial RNase P Protein 3), a protein-only RNase P (PRORP), is the nuclease component of the mtRNase P complex and requires a two-protein S-adenosyl methionine (SAM)-dependent methyltransferase MRPP1/2 sub-complex to function. Dysfunction of mtRNase P is linked to several human mitochondrial diseases, such as mitochondrial myopathies. Despite its central role in mitochondrial RNA processing, little is known about how the protein subunits of mtRNase P function synergistically. Here we use purified mtRNase P to demonstrate that mtRNase P recognizes, cleaves, and methylates some, but not all, mitochondrial pre-tRNAsin vitro. Additionally, mtRNase P does not process all mitochondrial pre-tRNAs uniformly, suggesting the possibility that some pre-tRNAs require additional factors to be cleavedin vivo. Consistent with this, we found that addition of the MRPP1 co-factor SAM enhances the ability of mtRNase P to bind and cleave some mitochondrial pre-tRNAs. Furthermore, the presence of MRPP3 can enhance the methylation activity of MRPP1/2. Taken together, our data demonstrate that the subunits of mtRNase P work together to efficiently recognize, process and methylate human mitochondrial pre-tRNAs.

In human, amyloid-beta (Aβ) and islet amyloid polypeptide (hIAPP) aggregations are linked to Alzheimer’s disease and Type-2 Diabetes, respectively. There is significant interest in better understanding the aggregation process by using chemical tools. Here, we show the ability of a cationic polymethacrylate-copolymer (PMAQA) to quickly induce β-hairpin structure and promote fibrillation in Aβ40, and to constrain the conformational plasticity of hIAPP for several days and inhibit its aggregation at sub-micromolar concentrations. NMR experiments and atomistic molecular dynamics simulations reveal that PMAQA electrostatically interacts with Aβ40’s Glu22 and Asp23 followed by β-sheet induction while it binds strongly to the closest proximity of amyloid core domain (NFGAIL) of hIAPP and restrain its structural rearrangement. This study provides a valuable approach to develop polymer-based anti-amyloid inhibitors that may diminish the population of intermediates of Aβ40 or hIAPP.

Protein prenylation involves the attachment of a hydrophobic isoprenoid moiety to the C-terminus of proteins. Several small GTPases, including members of the Ras and Rho subfamilies, require prenylation for their normal and pathological functions. Recent work has suggested that SmgGDS proteins regulate the prenylation of small GTPases in vivo. Using RhoA as a representative small GTPase, we directly test this hypothesis using biochemical assays and present a mechanism describing the mode of prenylation regulation. SmgGDS-607 completely inhibits RhoA prenylation catalyzed by protein geranylgeranyltransferase I (GGTase-I) in an in vitro radiolabel incorporation assay. SmgGDS-607 inhibits prenylation by binding to and blocking access to the C-terminal tail of the small GTPase (substrate sequestration mechanism) rather than via inhibition of the prenyltransferase activity. The reactivity of GGTase-I with RhoA is unaffected by addition of nucleotides. In contrast, the affinity of SmgGDS-607 for RhoA varies with the nucleotide bound to RhoA; SmgGDS-607 has a higher affinity for RhoA-GDP compared to RhoA-GTP. Consequently, the prenylation blocking function of SmgGDS-607 is regulated by the bound nucleotide. This work provides mechanistic insight into a novel pathway for the regulation of small GTPase protein prenylation by SmgGDS-607 and demonstrates that peptides are a good mimic for full-length proteins when measuring GGTase-I activity.

Ribonuclease P (RNase P) is an endonuclease that catalyzes the essential removal of the 5′ end of tRNA precursors. Until recently, all identified RNase P enzymes were a ribonucleoprotein with a conserved catalytic RNA component. However, the discovery of protein-only RNase P (PRORP) shifted this paradigm, affording a unique opportunity to compare mechanistic strategies used by naturally evolved protein and RNA-based enzymes that catalyze the same reaction. Here we investigate the enzymatic mechanism of pre-tRNA hydrolysis catalyzed by the NYN (Nedd4-BP1, YacP nuclease) metallonuclease of Arabidopsis thaliana, PRORP1. Multiple and single turnover kinetic data support a mechanism where a step at or before chemistry is rate-limiting and provide a kinetic framework to interpret the results of metal alteration, mutations, and pH dependence. Catalytic activity has a cooperative dependence on the magnesium concentration (nH = 2) under kcat/Km conditions, suggesting that PRORP1 catalysis is optimal with at least two active site metal ions, consistent with the crystal structure. Metal rescue of Asp-to-Ala mutations identified two aspartates important for enhancing metal ion affinity. The single turnover pH dependence of pre-tRNA cleavage revealed a single ionization (pKa ∼ 8.7) important for catalysis, consistent with deprotonation of a metal-bound water nucleophile. The pH and metal dependence mirrors that observed for the RNA-based RNase P, suggesting similar catalytic mechanisms. Thus, despite different macromolecular composition, the RNA and protein-based RNase P act as dynamic scaffolds for the binding and positioning of magnesium ions to catalyze phosphodiester bond hydrolysis. Background: A protein-only ribonuclease P (PRORP) has been recently discovered. Results: PRORP activity has a single ionization (pKa ∼ 8.7) important for catalysis and a cooperative dependence on Mg2+ (nH = 2). Conclusion: PRORP uses catalytic strategies similar to RNA-dependent RNase P. Significance: These results provide evidence for the mechanistic convergence of two different enzymatic macromolecules (RNA and protein) that perform the same biological function.

Mammalian mitochondrial tRNA(Ser(UCN)) (mt-tRNA(Ser)) and pyrrolysine tRNA (tRNA(Pyl)) fold to near-canonical three-dimensional structures despite having noncanonical secondary structures with shortened interhelical loops that disrupt the conserved tRNA tertiary interaction network. How these noncanonical tRNAs compensate for their loss of tertiary interactions remains unclear. Furthermore, in human mt-tRNA(Ser), lengthening the variable loop by the 7472insC mutation reduces mt-tRNA(Ser) concentration in vivo through poorly understood mechanisms and is strongly associated with diseases such as deafness and epilepsy. Using simulations of the TOPRNA coarse-grained model, we show that increased topological constraints encoded by the unique secondary structure of wild-type mt-tRNA(Ser) decrease the entropic cost of folding by ∼2.5 kcal/mol compared to canonical tRNA, offsetting its loss of tertiary interactions. Further simulations show that the pathogenic 7472insC mutation disrupts topological constraints and hence destabilizes the mutant mt-tRNA(Ser) by ∼0.6 kcal/mol relative to wild-type. UV melting experiments confirm that insertion mutations lower mt-tRNA(Ser) melting temperature by 6-9 °C and increase the folding free energy by 0.8-1.7 kcal/mol in a largely sequence- and salt-independent manner, in quantitative agreement with our simulation predictions. Our results show that topological constraints provide a quantitative framework for describing key aspects of RNA folding behavior and also provide the first evidence of a pathogenic mutation that is due to disruption of topological constraints.