Structural Studies of pH Effects on Botulinum Toxins A & E

Botulinum neurotoxins (BoNTs) are responsible for botulism, a paralytic disease which can be lethal if not treated in time. They act by entering neurons and targeting the SNARE proteins (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor), which in turn blocks neurotransmission. However, these toxins can be repurposed for therapeutic use to treat a large number of conditions. The most studied serotypes are A and E (BoNT/A and BoNT/E, respectively), with notable differences in duration of action and domain spatial organisation. It has been shown that these toxins only exert their activity if the pH drops to 5 or lower, but it is unclear what effect the pH environment has on the toxin which drives this. Currently, the only available structural information on BoNTs is from X-ray crystallography which fixes the protein into a rigid crystal lattice. This gives limited information on its flexible regions, and no information about its dynamics and solution behaviour. To gain insight into this, molecular dynamic (MD) simulations were conducted under varying pH conditions. For BoNT/E, these simulations revealed a shift in conformational populations in solvated systems at pH ≤ 5 when compared to simulations at pH > 5, with the protein adopting a more extended conformation in the former. This was confirmed by analytical ultra-centrifugation (AUC), while small-angle X-ray scattering (SAXS) validated the two major conformations observed in the MD simulations. For BoNT/A, a major conformational change was not observed, but a rare event was identified by MD (in 0.014% of frames studied) which may explain the longer onset of action compared to BoNT/E. Another key difference between the two structures of BoNT/E and BoNT/A is the large number of contacts between a conserved region termed the “switch” and the binding domain (BD) in BoNT/A, which are absent in BoNT/E

An Integrated Structural Mechanism for Relief of Autoinhibition and Membrane Targeting in Cytohesin Family Guanine Nucleotide Exchange Factors: A Dissertation

Guanine nucleotide exchange factors (GEFs) regulate and organize diverse cellular processes through their role in converting GTPases from the inactive GDP bound state to the active GTP bound state. An increasing number of GEFs undergo autoregulatory mechanisms through complex intramolecular interactions. Relief of autoinhibition involves specific phosphorylation or binding to lipid and/or effector proteins at sites distal from the catalytic domain, and is often coupled to membrane recruitment. In Cytohesin Arf GEFs, the catalytic Sec7 domain is autoinhibited by a linker region and C-terminal helix flanking a Pleckstrin Homology (PH) domain. Upon binding of the PH domain to low abundance phosphoinositides, the GTPase Arf6-GTP can both relieve autoinhibition and recruit Cytohesins to the plasma membrane. This thesis focuses on determining the molecular mechanism underlying both these functions. The structural mechanisms by which Arf6-GTP binding relieves autoinhibition were studied using biochemical and crystallographic studies. The crystal structure of the Grp1 PH domain in complex with Arf6 revealed that Arf6-GTP binding relieves autoinhibition through competitive sequestration of the inhibitory elements into grooves formed at the periphery of the interface. Importantly, the interaction orients all known membrane targeting components to a common surface. Detailed biochemical studies showed a common mode of binding among Cytohesin family members in which phosphoinositide head group binding primes the interaction with Arf6, and membrane recruitment of both stimulatory and substrate Arf enhances the effect. To assess changes in the Sec7 domain conformation upon activation, Size Exclusion Chromatography in line with Small Angle X-Ray Scattering (SEC-SAXS) was performed. The unique nature of this data led to the development of a novel data analysis and processing strategy. A graphically based, python-extensible software package was created for data normalization, buffer correction, Guinier Analysis, and constant background subtraction. As an unbiased substitute for traditional buffer subtraction, a method to reconstruct the protein scattering through singular value decomposition (SVD) and linear combination of the basis vectors was developed. These methods produced exceptional data quality and allowed versatility for application to other data collection techniques or systems, especially those lacking confident buffer matching or low signal. SEC-SAXS confirmed the overall structure of autoinhibited Grp1 in solution and showed only slight overall changes upon activation by deletion of the autoinhibitory Cterminal helix. Fusion of Arf6 with Grp1 produced a consistently elongated shape in the active state that was incompatible with the autoinhibited or theoretical active positions of the Sec7 domain. Monte Carlo and rigid body modeling using known structural domains revealed a requirement for Sec7-PH linker flexibility in addition to Sec7 domain mobility. These data support an integrated structural model whereby phosphoinositides and Arf-GTP support nucleotide exchange at membranes through allosteric activation, membrane recruitment, and large-scale rearrangement of the Sec7 domain. Overall, these findings offer insight into Cytohesin function that can be applied to assess relief of autoinhibition in the context of other GEFs and GTPases

Application of Computational Molecular Biophysics to Problems in Bacterial Chemotaxis

The combination of physics, biology, chemistry, and computer science constitutes the promising field of computational molecular biophysics. This field studies the molecular properties of DNA, protein lipids and biomolecules using computational methods. For this dissertation, I approached four problems involving the chemotaxis pathway, the set of proteins that function as the navigation system of bacteria and lower eukaryotes. In the first chapter, I used a special-purpose machine for molecular dynamics simulations, Anton, to simulate the signaling domain of the chemoreceptor in different signaling states for a total of 6 microseconds. Among other findings, this study provides enough evidence to propose a novel molecular mechanism for the kinase activation by the chemoreceptor and reconcile previously conflicting experimental data. In the second chapter, my molecular dynamics studies of the scaffold protein cheW reveals the existence and role of a conserved salt-bridge that stabilizes the relative position of the two binding sites in the chew surface: the chemoreceptor and the kinase. The results were further confirmed with NMR experiments performed with collaborators at the University of California in Santa Barbara, CA. In the third chapter, my colleagues and I investigate the quality of homology modeled structures with cheW protein as a benchmark. By subjecting the models to molecular dynamics and Monte Carlo simulations, we show that the homology models are snapshots of a larger ensemble of conformations very similar to the one generated by the experimental structures. In the fourth chapter, I use bioinformatics and basic mathematical modeling to predict the specific chemoreceptor(s) expressed in vivo and imaged with electron cryo tomography (ECT) by our collaborators at the California Institute of Technology. The study was essential to validate the argument that the hexagonal arrangement of transmembrane chemoreceptors is universal among bacteria, a major breakthrough in the field of chemotaxis. In summary, this thesis presents a collection of four works in the field of bacterial chemotaxis where either methods of physics or the quantitative approach of physicists were of fundamental importance for the success of the project

Calcium sensor proteins in hearing and sight. Biochemical investigation of diseases-associated variants.

Calcium is a cation which plays a pivotal role as second messenger, thus its concentration in cells needs to be finely regulated. Many systems work for that purpose, including Ca2+ sensor proteins, which undergo conformational changes upon Ca2+ coordination via EF-hands. Ca2+ sensors can be ubiquitous or tissue specific. Examples in this sense are represented by Guanylate Cyclase Activating Protein 1 (GCAP1) and Calcium- and Integrin-Binding Protein 2 (CIB2), involved in sight and hearing respectively. Missense point mutations in GCAP1 and CIB2 were found to be associated with genetic diseases characterized by retinal dystrophies and/or deafness. During my PhD, I focused my attention on the characterization of two point mutations namely p.Glu111Val (E111V) in GCAP1, leading to Cone/Rod dystrophy in an Italian family, and p.Glu64Asp (E64D) in CIB2, linked to Usher syndrome type 1J (USH1J), a rare disease characterized by the copresence of blindness and deafness. In particular, I spent the first part of the PhD investigating the role of CIB2 which is still under debate, finding that it is per se uncapable to work as a Ca2+ sensor under physiological conditions and that the conservative mutation linked to USH1J perturbs an allosteric communication between pseudo-EF1 and EF3, thus blocking the protein in an unfunctional conformation. Then, I characterized E111V GCAP1, finding that it is incapable of regulating its molecular target (Guanylate Cyclase), leading to a constitutive active enzyme and thus a progressively high concentrations of Ca2+ and cGMP in cells, which may explain the pathological phenotype. Looking for a potential therapeutic approach for Cone-Rod dystrophies, we found that the well-established Ca2+-relay model, explaining the gradual activation of Guanylate Cyclase by multiple GCAP molecules following gradual changes in intracellular Ca2+ concentrations, seems to be species-specific, since it apparently does not work in the same way in humans as in mouse and bovine photoreceptors. Finally, we identified a general method for the characterization of the interaction between a ubiquitous Ca2+ sensor protein (calmodulin) and inorganic CaF2 nanoparticles, suggesting their suitability as devices for nanomedicine applications

G-protein coupled receptors activation mechanism: from ligand binding to the transmission of the signal inside the cell

G-protein coupled receptors (GPCRs) are the largest family of pharmaceutical drug targets in the human genome and are modulated by a large variety of en- dogenous and synthetic ligands. GPCRs activation usually depends on agonist binding (except for receptors with basal activity), which stabilizes receptor con- formations and allow the requirement and activation of intracellular transducers. GPCRs are unique receptors and very well studied, since they play an important role in a great number of diseases. They interact with different type of ligands (such as light, peptides, proteins) and different partners in the intracellular part (such as G-proteins or β-arrestins). Based on homology and function GPCRs are divided in five classes: Class A or Rhodopsin, Class B1 or Secretin, Class B2 or Adhesion, Class C or Glutamate, Class F or Frizzled. What is still missing in the state of the art of these receptor, and in particular in Class A, is a global study on different binding cavities with divergent properties, with the aim to discover common binding characteristics, preserved during years of evolution. Gaining more knowledge on common features for ligand recognition shared among all the recep- tors may become crucial to deeply understand the mechanism used to transmit the signal into the cell. In the first step of this thesis we have used all the solved Class A receptors structures to analyze and find, if exist, a common way to transmit the signal inside the cell. We identified and validated ten positions shared between all the binding cavities and always involved in the interaction with ligands. We demonstrated that residues in these positions are conserved and have co-evolved together. In a second step, we used these positions to understand how ligands could be positioned in the binding cavities of three study cases: Muscarinic receptors, Kisspeptin receptors and the GPR3 receptor. We did not have any experimental information a priori. We used homology modeling and docking techniques for the first two cases, adding molecular dynamics simulations in the third case. All the predictions and suggestions from the computational point of view, turned out to be very successful. In particular for the GPR3 receptor we were able to identify and validate by alanine-scanning mutagenesis the role of three functionally relevant residues. The latter were correlated with the constitutive and agonist-stimulated adenylate cyclase activity of GPR3 receptor. Taken together, these results suggest an important role of computational structural biology and pave the way of strong collaborations between computational and experimental researches

HUMAN AROMATIC L-AMINO ACID DECARBOXYLASE: WHEN STRUCTURE AND MOBILITY DRIVE EFFICIENT CATALYSIS. IMPLICATIONS FOR AADC DEFICIENCY

L’enzima Decarbossilasi degli L-amino acidi aromatici (AADC) è responsabile della sintesi di due neurotrasmettitori essenziali: la dopamina e la serotonina. AADC deve la sua attività catalitica alla chimica del suo cofattore, il piridossale 5’-fosfato (PLP). La struttura cristallografica dell’enzima da mammifero (precisamente da maiale che ha il 90% di identità con l’enzima umano) nella sua forma olo venne risolta venti anni fa e tale risoluzione aprì la strada ad importanti studi strutturali. Dieci anni dopo venne pubblicata la struttura umana di AADC nella sua forma apo evidenziando quali cambiamenti conformazionali avvengono quando il PLP viene legato dall’enzima. Le strutture apo e olo AADC hanno avuto notevole importanza per la comprensione della patogenicità di varianti enzimatiche associate alla malattia chiamata ‘Deficit da AADC’ (AADCd, OMIM#608643). Questa malattia autosomica recessiva molto rara è dovuta prevalentemente a mutazioni missenso sul gene AADC. I pazienti affetti da AADCd mostrano un’amAromatic L-Amino Acid Decarboxylase (AADC) is the enzyme responsible for the synthesis of two essential neurotransmitter dopamine and serotonin from L-Dopa and L-hydroxytryptophan. AADC owes its specific catalytic activity to the chemistry of its cofactor, pyrydoxal-5’-phosphate (PLP). Almost 20 years ago, the crystal structure of a mammalian holoAADC (porcine, sharing 90% of sequence identity) was solved and the availability of its 3D structure paved the way to structural studies. Moreover, 10 years later, human apoAADC structure was published, shedding light on the conformational rearrangement occurring on the apo enzyme upon addition of PLP. Importantly, apo and holoAADC structures provided crucial insights for the comprehension of the pathogenicity of a number of AADC deficiency associated variants. AADC deficiency (OMIM#608643) is a rare autosomal recessive inborn disease due to missense mutations in the AADC gene. Patients bearing these mutations show mild to severe phenotypes, whose destiny is often fatal. Due to the rarity of the disease and to the heterogeneous response to the treatments, medications are not often satisfactory. In the past years, some efforts on human recombinant AADC pathogenic variants have tried to provide support to the research on AADC deficiency by means of biochemical and biophysical approaches determining the impact of the amino acid substitutions on the enzyme features. Here, a further contribution to the comprehension of the AADC deficiency is provided. The crystal structure of human holoAADC has been solved under different conditions, both in its native and ligand bound form. The combination of crystallographic studies, molecular dynamics simulations (MD) and site directed mutagenesis uncovered novel aspects of the AADC structure-function relationship. Moreover, the characterization of 21 novel identified pathogenic variants (spread on each AADC domain, N-terminal, Large and C-terminal Domains) led to the widening of the range of enzymatic phenotypes associated to AADC deficiency. The proposed combination of biochemical and kinetic studies permitted to determine correlations between structural and functional signals. Enzymatic phenotypes span from variants characterized by a mild phenotypes to variants (mainly located at the NTD-CTD interface) whose dramatic structural defects lead to a catalytic incompetence. In addition, MD simulations and in solutions data point out a critical role for the loop3 element that contains the essential catalytic residue Tyr332. A group of variants affecting loop3 has been identified as catalytically incompetent and their structural features have been dissected thanks also to the solving of the crystal structure of pathogenic variant L353P, which constitutes the first solved structure of an AADC variant. Altogether, this study on human AADC provides new elements for the comprehension of the structure-function relationship of AADC with a particular focus on protein dynamics and mobility. Lastly, structural details might represent the basis for both the designing of novel specific inhibitors and for a better comprehension of the molecular aspects of the variants associated with the AADC deficiency

Structural and biochemical characterization of ribosome small subunit-dependent GTPase A (RsgA) from Pseudomonas aeruginosa

The increase in antibiotic resistance among pathogenic bacterial strains presents a significant health threat. The main efforts to combat antibiotic resistance are focused on the development of new antibiotics targeting protein biosynthesis. Ribosome, the large molecular machine responsible for this process, and proteins involved in the translational process represent ideal targets of molecules with antibacterial activity. The ribosome assembly in vivo is an intricate and finely tuned process promoted by the action of several proteins acting as assembly factors, whose precise role is still largely unknown. Small GTPases represent the largest class of ribosome assembly factors in bacteria and are emerging as possible targets to be explored for the development of novel antibacterial strategies. Among them, of particular interest is the Ribosome small subunit-dependent GTPase A (RsgA). RsgA is a late-stage ribosome biogenesis factor involved in the 30S subunit maturation, broadly conserved among bacteria but absent in eukaryotes. RsgA is a circulary permutated GTPase that belongs to an interesting class of GTPases, termed HAS-GTPase, that lack the conserved catalytic glutamine. The circularly permutated GTP binding site is flanked by an OB-fold domain at the N-terminus and by a zinc binding domain at the C-terminus. Despite the large amount of biochemical, structural and genetic data on RsgA achieved in the last decade, its mechanism of action is still not completely understood. Here we focus on the structural and functional characterisation of RsgA from the human pathogenic bacterium Pseudomonas aeruginosa (PaRsgA). The main goal of this work is the determination of the PaRsgA structure by X-ray crystallography. To date, no structure is available for RsgA from this opportunistic pathogen. This knowledge will allow investigate the molecular features for the recognition of GDP and GTP as well as the key determinants for the mechanism of GTP hydrolysis. Moreover, an accurate kinetic analysis of PaRsgA interaction with GDP and GTP, together with a detailed functional characterization of PaRsgA, provided the determination of substrates affinity and biochemical parameters of GTP hydrolysis. The results obtained will pave the way for future experiments aimed at the characterization of the binding mechanism underlying ribosome recognition and to get key insight the GTPase activity of PaRsgA in the presence of other assembly factors and/or the ribosomal particle

Chemical Biology, Biochemical and Structural Studies of MDN1, an AAA Protein Required for Ribosome Biogenesis

Cellular proteins are synthesized by ribosomes, which are ~3 MDa macromolecular complexes comprised of four ribosomal RNAs and ~80 ribosomal proteins in yeast. The biogenesis of such complicated ribonucleoprotein complexes is a highly regulated, multistep process requiring a plethora of more than 200 unique assembly factors. Energy-harnessing enzymes, such as ATPases and GTPases, are needed to remodel the precursors of ribosomes at fast time scales. Mdn1 is an essential dynein-like AAA protein (ATPases Associated with various Activities) that releases specific assembly factors from the precursors of 60S subunit of ribosomes. However, Mdn1’s unusually large size (~5000 amino acids in a single polypeptide) and the transient nature of intermediates of ribosome biogenesis have limited our understanding how Mdn1 remodels pre-60S particles. In addition, the limited homology of Mdn1 to other well-studied proteins, including dyneins, has restricted our understanding of its function. Here, I first combined chemical and biochemical approaches to develop and validate ribozinoindoles (Rbins) as the cell-permeable inhibitors of Mdn1, which are the first potent and selective inhibitors of ribosome biogenesis in eukaryotes. These compounds can be further used to dissect the dynamic functions of Mdn1 during the multistep process of ribosome biogenesis. In addition, I solved three cryo-EM structures of both full-length and truncated Mdn1 (resolution up to 4.0 Å) that provided the first pseudo-atomic models for Mdn1 in two distinct nucleotide states. Remarkably, Mdn1’s the C-terminal MIDAS domain (Metal Ion-Dependent Adhesion Site), which interacts with other ribosome assembly factors, docks onto the N-terminal AAA ring in a nucleotide state-specific manner, even though they are separated by more than 2000 aa. These data suggest that conformational changes in the AAA ring can be directly transmitted to the MIDAS domain, thereby driving the selective release of the MIDAS-bound assembly factors from the precursors of 60S subunit of ribosomes. Together, these chemical biology, biochemical and structural studies of Mdn1 reveal how an AAA protein can contribute to the dynamic ribosome biogenesis process in eukaryotes

Nucleotide and Polymerization Effects on Actin Structure and Dynamics

Actin is one of the most highly conserved and abundant proteins found in eukaryotic cells, essential for determining cell shape, polarity and motility, as well as protein transport and even mitosis and muscle contraction. These functions depend on actin’s ability to exist – under the strict control of nucleotide hydrolysis and interaction with actin binding partners – as either a monomer (G-actin) or a polymer (F-actin). Over the last seven decades of research, much has been determined in the way of actin function, but key questions still remain. First, the affinity of actin for the >150 actin binding proteins, and for polymerization, is reliant on upon actin’s nucleotide state but the structural changes that occur upon nucleotide hydrolysis are not so clear. Next, the two ends of the actin filament have different properties, with incoming ATP-actin subunits preferentially adding to the barbed end of the filament and ADP-actin subunits preferentially dissociating from the pointed end. Much like with the structural changes that occur upon hydrolysis, the physical and biochemical bases for these differences are unknown. Finally, as the cell relies on the strict regulation of the actin filament, alterations to actins sequence are poorly tolerated. Although many mutations prove to be lethal to the organism, over 140 disease causing actin mutants have been reported, with a large subset clustering on actin’s pathogenic helix (residues 113-125). Little is known about the structural consequences of these mutations and how they relate to disease. In my thesis, I use molecular dynamics simulations of both G- and F-actin to probe these questions. Actin itself has two clefts, the nucleotide-binding cleft at the center of the protein, and the target-binding cleft at the bottom of the protein between subdomains 1 and 3, where the majority of actin binding proteins dock. I show that changes within the nucleotide-binding cleft propagate down to the target-binding cleft through the intermediary C-terminal hinge (A331-Y337). Within the target-binding cleft itself, I identify a new loop at the profilin binding site (FQQ-loop: S348-W356) that moves by nearly 5 Å in the ATP state to partially obstruct the target-binding cleft. All of these changes help explain nucleotide state specificity for actin binding proteins. My work also reveals that ATP G-actin takes on a flatter conformation that is structurally similar to F-actin’s barbed end protomer, explaining the observation that ATP G-actin polymerizes faster than its ADP counterpart. I find that the pointed end of the filament takes on a conformation that is divergent from remainder of the filament and monomer simulations, effectively raising the conformational energy barrier for the addition of actin protomers. I also looked at the structural consequences of the deafness causing mutations K118M/N. The mutations to K118 result in changes in the structure and dynamics of the D-loop, alterations in the structure of the H73-loop as well as the sidechain orientations of W79 and W86, changes in nucleotide exchange rates, and significant shifts in the twist of the actin monomer. With K118N the twist of the monomer is nearly identical to the F-actin protomer, and in vitro polymerization assays show that this mutation results in faster polymerization. Taken together, it is evident that mutations at this site give rise to a series of small changes that can be tolerated in vivo, but result in misregulation of actin assembly and dynamics.PHDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147698/1/ljepsen_1.pd

Multiscale Modeling of Familial Cardiomyopathy-linked Tropomyosin Mutations

Mutations in proteins of the cardiac sarcomere can alter muscle function, leading to a hypercontractile or a hypocontractile state of the heart. These mutational insults can also lead to disease states over time, such as hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM). One such gene, TPM1, which encodes the sarcomeric regulatory protein α-tropomyosin, has been linked to cases of both familial HCM and DCM. However, it remains unclear how different mutations to the same gene can cause different pathogenic phenotypes. Without clear predictive genotype-phenotype relationships, the value of clinical genetic testing in screening and treating families is inherently limited. This work focuses on the development of two tools to improve understanding of the connection between genotype and phenotype for sarcomeric genes: 1) a computational model of the cardiac thin filament and 2) an engineered tissue model capable of expressing arbitrary mutations through use of adenoviral transduction. By pairing these tools with other complementary methodologies (molecular dynamics and in vitro motility assays [IVMA]) we seek to demonstrate that they can form the basis of accurate classification of TPM1 variants of unknown significance (VUS) into HCM or DCM phenotypes. We first designed and implemented a Markov chain-Monte Carlo model for simulating thin filament activation. We wanted to produce a detailed model that was capable of predicting both steady-state and dynamic force production while incorporating detailed mechanisms of regulation. To do this, we investigated the regulatory mechanism of the sarcomeric protein troponin I (TnI). It was long thought the inhibitory peptide domain (IP) of TnI acted as the sole actin-binding region that holds tropomyosin in the myosin-blocking position. More recently, evidence has arisen that the C-terminal mobile domain (MD) of TnI also binds actin and may also contribute to this inhibition. To properly incorporate these findings, we created both a 16-state model with TnI-IP as the sole regulatory domain and a 24-state TnI-IP+MD version. Comparison of these models showed that assumption of a second actin-binding site allows the individual domains to have a lower affinity for actin than with IP acting alone. We also tested the 24-state model’s ability to represent steady-state experimental data in the case of disruption of either the IP or MD and we were able to capture qualitative changes in several properties as seen in experimental data. Overall, our analyses support a paradigm in which two domains of TnI bind with moderate affinity to actin, working in tandem to regulation the thin filament. To begin the characterization of mutations to TPM1, molecular dynamics simulations were used to predict important structural and mechanical changes. We applied this to two mutants: the DCM-linked M8R and the HCM-linked S215L. M8R increased flexibility of the tropomyosin chain and enhanced affinity for the blocked or inactive state of tropomyosin on actin. S215L also increased flexibility of the tropomyosin chain while enhancing affinity for the closed state of tropomyosin on actin in which myosin binding sites are revealed. Applying these molecular effects to the 24-state Markov model reproduced the shifts in calcium sensitivity, maximum force, and cooperativity that were also observed in IVMA experiments. The model was then used to simulate the impact of M8R or S215L expression on twitch behavior. These dynamic simulations predicted that M8R would reduce peak force and duration of contraction in a dose-dependent manner. To evaluate this prediction, TPM1 M8R was expressed via adenovirus in engineered heart tissues and isometric twitch force was observed. The mutant tissues showed depressed contractility and twitch duration that agreed in detail with model predictions. For S215L, simulations predicted a hypercontractile twitch phenotype. Mechanical testing of genetically engineered tissues homozygous for mutant S215L TPM1 also showed an increase in peak force and slowed relaxation when compared to isogenic WT tissues. In the final study, we characterized four TPM1 VUS using a combination of molecular modeling, IVMA, and engineered tissue. First, 20 candidate VUS were analyzed computationally using molecular dynamics and energy minimization calculations to predict each variant’s effects on TPM1 structure and association with thin filament proteins. From this analysis, four variants representing a spectrum from most to least predicted pathogenicity (A102D, D258E, K233N, and A239T) were selected for further study. Predictions were tested for each variant via engineered heart tissues. Mechanical testing of the tissues revealed an HCM phenotype for A102D and D258E, but a DCM phenotype for A239T and K233N. The pathogenic phenotypes of these selected variants reveals robust progress toward our long-term goal of computational prediction of disease risk for novel TPM1 variants