Biology subjects

Engen, J. R.

Publications and source records attributed to Engen, J. R..

18 recordsLinked to original sources

bioRxiv2026

Magnesium as a Conformational Gatekeeper of KRAS: Structural Dynamics and Therapeutic Implications

Magnesium serves as an essential cofactor for small GTPases, yet its structural role in regulating KRAS conformational dynamics and nucleotide exchange remains poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS), native mass spectrometry, and functional assays to elucidate how Mg2+ stabilizes the KRAS conformational ensemble and constrains transitions between GDP- and GTP-bound states. Depletion of Mg2+ triggers widespread increases in structural dynamics throughout KRAS--spanning the p-loop, 1-helix, switch I, nucleotide-binding region, and distal helices--revealing a global loosening of the protein fold that favors an open, nucleotide exchange-competent state. Mg2+ titration experiments demonstrate that individual structural elements exhibit distinct Mg2+ dependencies: the p-loop and 1-helix recover native dynamics at micromolar concentrations, whereas switch I requires millimolar levels, underscoring its exceptionally high sensitivity to Mg2+ for structural stabilization. KRAS bound to the catalytic domain of exchange factor SOS1 displays an HDX signature closely resembling the Mg2+-free state, indicating that SOS1 promotes nucleotide exchange by transiently perturbing Mg2+ coordination while simultaneously stabilizing switch I. Consistently, phosphomimetic KRAS S17E variant, which disrupts a critical Mg2+-coordinating residue, exhibits pronounced global destabilization--reinforcing the central importance of Mg2+ in maintaining structural integrity. Taken together our findings show that Mg2+ acts as a master regulator of KRAS structural dynamics and reveal Mg2+-sensitive hotspots that might represent promising targets for next-generation KRAS therapeutics.

biochemistry↗

bioRxiv2026

How the non-motile kinesin KIF7 adapts conserved kinesin principles for itsfunction in Hedgehog signaling

KIF7 is an atypical, non-motile kinesin that regulates Hedgehog signaling by concentrating GLI transcription factors at the cilium tip. How canonical kinesin principles for intracellular transport are repurposed to support KIF7s function as a signaling scaffold remains unclear. KIF7 exists in an autoinhibited state that is relieved by GLI binding, promoting microtubule association. We examined this regulatory mechanism by combining HDX-MS and AlphaFold modeling of a minimal KIF7 dimer, both alone and in complex with the GLI2 zinc-finger domain. Our HDX-MS data indicate that the highly negatively charged neck-coil dimerization domain of KIF7, which serves as the GLI2-binding site, is intramolecularly protected in the absence of GLI2. Consistent with this, AlphaFold models suggest that the motor domain folds back onto the neck-coil via KIF7s unusually long neck-linker, sterically occluding the microtubule-binding interface. This occurs through a mechanism conceptually analogous to, but structurally distinct from, autoinhibition in motile kinesins. GLI2 binding to the KIF7 neck-coil displaces the motor domain and induces allosteric changes that propagate to the microtubule-binding surface, thereby activating microtubule binding. ATP turnover further modulates KIF7s microtubule binding-unbinding equilibrium. Together, these findings reveal how a kinesin is adapted for a non-motile function as a scaffold in Hedgehog signaling.

biochemistry↗

bioRxiv2026

Thermodynamic Insights into the WT and Y220C TP53 DBDs Reveals that the Oncogenic Y220C Variant is a Loss of Function Mutation for Zn2+-binding at Physiological Temperature

We present evidence of previously unrecognized allosteric connectivity across the TP53 DNA binding domain (DBD). Specifically, we have found evidence of explicit influence on the Zn2+-binding site from the region surrounding the hotspot Y220C mutation. This allosteric connectivity is intertwined with a temperature-dependent destabilization of Zn2+ binding in both the WT and Y220C DBDs. Our studies indicate that the Y220C mutation exacerbates this temperature-dependent destabilization of Zn2+-binding to result in overall destabilization of the Y220C variant. We provide detailed thermodynamic evidence that Rezatapopt, a small molecule reactivator of the Y220C DBD, engages Y220C through two distinct thermodynamic pathways and restores WT-level Zn2+-affinity to this oncogenic variant. A series of thermodynamic models describing the WT and Y220C conformational landscapes, as well as the Rezatapopt binding mechanisms, are proposed.

biochemistry↗

bioRxiv2026

Small molecule stabilization of diverse amyloidogenic immunoglobulin light chains revealed by hydrogen-deuterium exchange mass spectrometry

Immunoglobulin light chains, a component of antibodies, can misfold and aggregate to cause systemic AL amyloidosis. Aggregation, including amyloid fibril formation, requires unfolding of the full-length light chain from its native state, and in most cases aberrant proteolysis. Small molecules that bind to the native state of light chains to stabilize them against conformational excursions and proteolysis are under development as drug candidates for AL amyloidosis. Since each patient has a unique light chain sequence, a challenge for candidate stabilizer drugs is to bind multiple light chains and suppress their dynamics. Here, we used hydrogen-deuterium exchange measured by mass spectrometry to characterize the binding of six small molecule stabilizers to eleven different {lambda} light chain proteins. Despite structural and dynamic differences among the light chains, the binding of the most efficacious stabilizer molecule led to increased protection from hydrogen exchange, consistent with reduced local and global unfolding. Protection upon binding was most prominent in residues within complementarity determining region 3 and framework region 4 of the light chain variable domains, which undergo major conformational changes enabling amyloid formation. Stabilizer binding also reduced the rate at which all light chains were cleaved by protease. These data show that these stabilizers suppress the range of conformational dynamics associated with light chain aggregation, supporting their therapeutic potential. HighlightsO_LISmall-molecule kinetic stabilizers suppress conformational dynamics and aberrant proteolysis of diverse amyloidogenic full-length {lambda} light chains. C_LIO_LIHDX-MS indicates conserved protection in regions forming the interface between the two variable domains of the light chain dimer, corresponding to residues that form the core of patient-derived amyloid fibrils. C_LIO_LIPLIMSTEX binding titrations show coupled binding, dimerization and stabilization at sub-micromolar kinetic stabilizer concentrations. C_LIO_LISmall molecule kinetic stabilization across different {lambda} sequences supports kinetic stabilizers as a promising therapeutic strategy for AL amyloidosis. C_LI

biophysics↗

bioRxiv2024

An SH3-binding allosteric modulator stabilizes the global conformation of the AML-associated Src-family kinase, Hck

While ATP-site inhibitors for protein-tyrosine kinases are often effective drugs, their clinical utility can be limited by off-target activity and acquired resistance mutations due to the conserved nature of the ATP-binding site. However, combining ATP-site and allosteric kinase inhibitors can overcome these shortcomings in a double-drugging framework. Here we explored the allosteric effects of two pyrimidine diamines, PDA1 and PDA2, on the conformational dynamics and activity of the Src-family tyrosine kinase Hck, a promising drug target for acute myeloid leukemia. Using 1H-15N HSQC NMR, we mapped the binding site for both analogs to the SH3 domain. Despite the shared binding site, PDA1 and PDA2 had opposing effects on near-full-length Hck dynamics by hydrogen-deuterium exchange mass spectrometry, with PDA1 stabilizing and PDA2 disrupting the overall kinase conformation. Kinase activity assays were consistent with these observations, with PDA2 enhancing kinase activity while PDA1 was without effect. Molecular dynamics simulations predicted selective bridging of the kinase domain N-lobe and SH3 domain by PDA1, a mechanism of allosteric stabilization supported by site-directed mutagenesis of N-lobe contact sites. Cellular thermal shift assays confirmed SH3 domaindependent interaction of PDA1 with wild-type Hck in myeloid leukemia cells and with a kinase domain gatekeeper mutant (T338M). These results identify PDA1 as a starting point for Src-family kinase allosteric inhibitor development that may work in concert with ATP-site inhibitors to suppress the evolution of resistance.

pharmacology and toxicology↗

bioRxiv2023

Impact of the clinically approved BTK inhibitors on the conformation of full-length BTK and analysis of the development of BTK resistance mutations in chronic lymphocytic leukemia.

Inhibition of Brutons tyrosine kinase (BTK) has proven to be highly effective in the treatment of B-cell malignancies such as chronic lymphocytic leukemia (CLL), autoimmune disorders and multiple sclerosis. Since the approval of the first BTK inhibitor (BTKi), Ibrutinib, several other inhibitors including Acalabrutinib, Zanubrutinib, Tirabrutinib and Pirtobrutinib have been clinically approved. All are covalent active site inhibitors, with the exception of the reversible active site inhibitor Pirtobrutinib. The large number of available inhibitors for the BTK target creates challenges in choosing the most appropriate BTKi for treatment. Side-by-side comparisons in CLL have shown that different inhibitors may differ in their treatment efficacy. Moreover, the nature of the resistance mutations that arise in patients appears to depend on the specific BTKi administered. We have previously shown that Ibrutinib binding to the kinase active site causes unanticipated long-range effects on the global conformation of BTK (Joseph, R.E., et al., 2020, https://doi.org/10.7554/eLife.60470). Here we show that binding of each of the five approved BTKi to the kinase active site brings about distinct allosteric changes that alter the conformational equilibrium of full-length BTK. Additionally, we provide an explanation for the resistance mutation bias observed in CLL patients treated with different BTKi and characterize the mechanism of action of two common resistance mutations: BTK T474I and L528W.

biochemistry↗

bioRxiv2023

Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states

Full-length BTK has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology (PHTH) domain and proline-rich regions (PRR) contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveals only the SH3-SH2-kinase core with no electron density visible for the PHTH-PRR segment. CryoEM data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH-PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3-SH2-kinase core. On the way to activation, disassembly of the SH3-SH2-kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with PIP 3. Membrane-induced dimerization activates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to trans-autophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.

biophysics↗

bioRxiv2023

Role of Complementarity-Determining Regions 1 and 3 in Pathologic Amyloid Formation by Human Immunoglobulin κ1 Light Chains

Immunoglobulin light chain (LC) amyloidosis is a life-threatening disease whose understanding and treatment is complicated by vast numbers of patient-specific mutations. To address molecular origins of the disease, we explored 14 patient-derived and engineered proteins related to {kappa}1-family germline genes IGKVLD-33*01 and IGKVLD-39*01. Hydrogen-deuterium exchange mass spectrometry analysis of local conformational dynamics in full-length recombinant LCs and their fragments was integrated with studies of thermal stability, proteolytic susceptibility, amyloid formation, and amyloidogenic sequence propensities using spectroscopic, electron microscopic and bioinformatics tools. The results were mapped on the atomic structures of native and fibrillary proteins. Proteins from two {kappa}1 subfamilies showed unexpected differences. Compared to their germline counterparts, amyloid LC related to IGKVLD-33*01 was less stable and formed amyloid faster, whereas amyloid LC related to IGKVLD-39*01 had similar stability and formed amyloid slower. These and other differences suggest different major factors influencing amyloid formation. In 33*01-related amyloid LC, these factors involved mutation-induced destabilization of the native structure and probable stabilization of amyloid. The atypical behaviour of 39*01-related amyloid LC tracked back to increased dynamics/exposure of amyloidogenic segments in {beta}CV and {beta}EV that could initiate aggregation, combined with decreased dynamics/exposure near the Cys23-Cys88 disulfide whose rearrangement is rate-limiting to amyloidogenesis. The results suggest distinct amyloidogenic pathways for closely related LCs and point to the antigen-binding, complementarity-determining regions CDR1 and CDR3, which are linked via the conserved internal disulfide, as key factors in amyloid formation by various LCs.

biochemistry↗

bioRxiv2022

Increase the flow rate and improve hydrogen deuterium exchange mass spectrometry

Reversed-phase peptide separation in hydrogen deuterium exchange (HDX) mass spectrometry (MS) must be done with conditions where the back exchange is the slowest possible, the so-called quench conditions of low pH and low temperature. To retain maximum deuterium, separation must also be done as quickly as possible. The low temperature (0 {degrees}C) of quench conditions complicates the separation and leads primarily to a reduction in separation quality and an increase in chromatographic backpressure. To improve the separation in HDX MS, one could use a longer gradient, smaller particles, a different separation mechanism (for example, capillary electrophoresis), or multi-dimensional separations such as combining ion mobility separation with reversed-phase separation. Another way to improve separations under HDX MS quench conditions is to use a higher flow rate where separation efficiency at 0 {degrees}C is more ideal. Higher flow rates, however, require chromatographic systems (both pumps and fittings) with higher backpressure limits. We tested what improvements could be realized with a commercial UPLC/UHPLC system capable of ~20,000 psi backpressure. We found that a maximum flow rate of 225 L/min (using a 1x50mm column packed with 1.8 m particles) was possible and that higher flow rate clearly led to higher peak capacity. HDX MS analysis of both simple and particularly complex samples improved, permitting both shorter separation time, if desired, and providing more deuterium recovery.

biophysics↗

bioRxiv2022

Resolving chaperone-assisted protein folding on the ribosome at the peptide level

The cellular environment is critical for efficient protein maturation, but how proteins fold during biogenesis remains poorly understood. We used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. On the ribosome, the nascent polypeptide folds via structured intermediates not populated during refolding from denaturant. Association with the ribosome allows these intermediates to form, as otherwise destabilizing C-terminal sequences remain confined in the ribosome exit tunnel. We find that partially-folded nascent chains recruit the chaperone Trigger factor, which uses a large composite hydrophobic/hydrophilic interface to engage folding intermediates without disrupting their structure. In addition, we comprehensively mapped dynamic interactions between the nascent chain and ribosomal proteins, tracing the path of the emerging polypeptide during synthesis. Our work provides a high-resolution description of de novo protein folding dynamics, thereby revealing new mechanisms by which cellular factors shape the conformational search for the native state.

biochemistry↗

bioRxiv2022

Von Willebrand factor A1 domain affinity for GPIbα and stability are differentially regulated by its O-glycosylated N-linker and C-linker

Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor to GPIb on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIb, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIb, which is induced by tensile force physiologically and regulated allosterically by the N-linker. Impact StatementBoth the polypeptide and attached O-glycans N-terminal to the A1 domain in von Willebrand factor lower its affinity for its ligand GPIb on platelets, its stability, and structural dynamics and decrease population of a high-affinity, intermediate state in unfolding. Key pointsO_LILigand-binding affinity of A1 and the stability of its native state is regulated by an N-terminal interdomain, O-glycosylated linker C_LI

biophysics↗

bioRxiv2021

Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains, rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C-terminus through the central pore of the hexameric double-ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.

biochemistry↗

bioRxiv2021

Allosteric control of Ubp6 and the proteasome via a bidirectional switch

The proteasome is the principal cellular protease, and recognizes target proteins that have been covalently marked by ubiquitin chains. The ubiquitin signal is subject to rapid editing at the proteasome, allowing it to reject substrates based on topological features of their attached ubiquitin chains. Editing is mediated by a key regulator of the proteasome, deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome-both by deubiquitinating proteasome-bound ubiquitin conjugates, and through a noncatalytic effect that does not involve deubiquitination. We report mutants in both Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations fall within its ILR element, defined here, which is conserved from yeast to mammals. The ILR is a component of the BL1 blocking loop, other parts of which obstruct ubiquitin access to the catalytic groove in free Ubp6. Rpt1 docking at the ILR opens the catalytic groove by rearranging not only BL1 but also a novel network of three directly interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition by Ubp6 are linked in that they were eliminated by the same Ubp6 and Rpt1 mutations. Ubp6 and ubiquitin together drive the proteasome into a unique conformational state associated with proteasome inhibition. Our results identify a multicomponent allosteric switch that exerts simultaneous control over the activity of both Ubp6 and the proteasome, and suggest that their active states are in general mutually exclusive. The findings lead to a new paradigm for allosteric control of deubiquitinating enzymes.

biochemistry↗

bioRxiv2021

Protection of the prodomain 1-helix correlates with latency in the transforming growth factor- family

The 33 members of the transforming growth factor beta (TGF-{beta}) family are fundamentally important for organismal development and homeostasis. Family members are synthesized and secreted as pro-complexes of prodomains that are non-covalently bound to the growth factor (GF). The pro-complexes of some members are latent and require activation steps to release the GF for signaling. Why some members are latent while others are non-latent is incompletely understood, but crystal structures and hydrogen-deuterium exchange (HDX) of four family members have begun to unravel how latency is regulated. Here, we extend this understanding by comparing pro-complex conformation in negative stain EM (nsEM) and HDX of ActA, BMP7, BMP9, BMP10, GDF8, TGF-{beta}1, and TGF-{beta}2. nsEM revealed that family members varied in either adopting cross-armed, open-armed, or V-armed configurations. Latency was achieved in both cross-armed and V-armed but not open-armed conformations. HDX revealed remarkably varying patterns of exchange between family members, consistent with large prodomain sequence divergence. We observed a strong correlation between latency and protection of the prodomain 1-helix from exchange, which in latent members coincided with greater buried surface area of the 1-helix and more hydrogen and cation-pi bonds from the prodomain fastener and GF to the 1-helix. Strong sequence conservation of the 1-helix and fastener only in latent members suggests that similar interactions are conserved and sufficient to confer latency. Moreover, most members exhibited rapid exchange in the unstructured "association region" at the prodomain N-terminus, highlighting their availability for interacting with factors that may regulate latency and extracellular storage.

biophysics↗

bioRxiv2020

Crystal Structure of the Werner's Syndrome Helicase

Werner syndrome helicase (WRN) plays important roles in multiple pathways of DNA repair and the maintenance of genome integrity. Recently, loss of WRN was identified as a strong synthetic lethal interaction for microsatellite instable (MSI) cancers making WRN a promising drug target. Yet, structural information for the helicase domain is lacking, which prevents structure-based design of drug molecules. In this study, we show that ATP binding and hydrolysis in the helicase domain are required for genome integrity and viability of MSI cancer cells. We then determined the crystal structure of an ADP bound form of the WRN helicase core at 2.2 [A] resolution. The structure features an atypical mode of nucleotide binding with extensive contacts formed by motif VI, which in turn defines the relative positioning of the two RecA like domains. The structure features a novel additional {beta}-hairpin in the second RecA and an unusual helical hairpin in the Zn2+ binding domain, and modelling DNA substrates based on existing RecQ DNA complexes suggests roles for these features in the binding of alternative DNA structures. We have further analysed possible interfaces formed from the interactions between the HRDC domain and the helicase core by NMR. Together, this study will facilitate the structure-based design of inhibitors against WRN helicase.

biochemistry↗

bioRxiv2018

Mitochondrial ClpX activates an essential metabolic enzyme through partial unfolding

Mitochondria can control the activity, quality, and lifetime of their proteins with their autonomous system of chaperones, but the signals that direct substrate-chaperone interaction and outcome are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting incorporation of cofactor. Here, we ask how unfolding by mtClpX directs activation. We identified sequence and structural features in ALAS that position mtClpX and provide a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding was limited to a subdomain extending from the mtClpX-binding site to the active site. Unfolding along this path was required for mtClpX to gate cofactor access to the ALAS active site. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and suggests how substrate-chaperone interactions can direct the outcome of remodeling.

biochemistry↗

bioRxiv2018

Fusion surface structure, function, and dynamics of gamete fusogen HAP2

HAP2 is a class II gamete fusogen in many eukaryotic kingdoms. A crystal structure of Chlamydomonas HAP2 shows a trimeric fusion state. Domains D1, D2.1 and D2.2 line the 3-fold axis; D3 and a stem pack against the outer surface. Surprisingly, hydrogen-deuterium exchange shows that surfaces of D1, D2.2 and D3 closest to the 3-fold axis are more dynamic than exposed surfaces. Three fusion helices in the fusion loop of each monomer expose hydrophobic residues at the trimer apex that are splayed from the 3-fold axis, leaving a solvent-filled cavity between the fusion loops in each monomer. At the base of the long fusion loop, Arg185 docks in a carbonyl cage. Comparisons to other structures, dynamics, and the greater effect on Chlamydomonas gamete fusion of mutation of axis-proximal than axis-distal fusion helices suggest that the apical portion of each monomer could tilt toward the 3-fold axis with merger of the fusion helices into a common fusion surface.

biochemistry↗

bioRxiv2017

Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation

Growth differentiation factor 8 (GDF8)/Myostatin is a latent TGF{-}{beta} family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid{-}cleaved GDF8 pro{-}complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro{-}complexes reveals a V{-}shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring{-}like, cross{-}armed conformation of latent TGF{-}{beta}1. Surprisingly, Tolloid{-}cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-{beta}1 to the 1-helix, latency lasso, and {beta}1 strand in the prodomain and to the {beta}6-7 strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.

biochemistry↗