Source code and structural PDBs that went into the Prediction of ternary complex structures using molecular dynamics and proteomics paper.

Targeted protein degradation (TPD) is a promising approach in drug discovery for degrading proteins implicated in diseases. A key step in this process is the formation of a ternary complex where a heterobifunctional molecule induces proximity of an E3 ligase to a protein of interest (POI), thus facilitating ubiquitin transfer to the POI. In this work, we characterize 3 steps in the TPD process. (1) We simulate the ternary complex formation of SMARCA2 bromodomain and VHL E3 ligase by combining hydrogen-deuterium exchange mass spectrometry with weighted ensemble molecular dynamics (MD). (2) We characterize the conformational heterogeneity of the ternary complex using Hamiltonian replica exchange simulations and small-angle X-ray scattering. (3) We assess the ubiquitination of the POI in the context of the full Cullin-RING Ligase, confirming experimental ubiquitinomics results. Differences in degradation efficiency can be explained by the proximity of lysine residues on the POI relative to ubiquitin. The formation of ternary degrader-protein complexes is a key step in the targeted degradation of proteins of interest. Here, the authors explore the structure and dynamics of such complexes applying high-performance computer simulations augmented with experimental data.

Targeted protein degradation (TPD) has emerged as a powerful approach in drug discovery for removing (rather than inhibiting) proteins implicated in diseases. A key step in this approach is the formation of an induced proximity complex, where a degrader molecule recruits an E3 ligase to the protein of interest (POI), facilitating the transfer of ubiquitin to the POI and initiating the proteasomal degradation process. Here, we address three critical aspects of the TPD process: 1) formation of the ternary complex induced by a degrader molecule, 2) conformational heterogeneity of the ternary complex, and 3) assessment of ubiquitination propensity via the full Cullin Ring Ligase (CRL) macromolecular assembly. The novel approach presented here combines experimental biophysical data—in this case hydrogen-deuterium exchange mass spectrometry (HDX-MS, which measures the solvent exposure of protein residues)—with all-atom explicit solvent molecular dynamics (MD) simulations aided by enhanced sampling techniques to predict structural ensembles of ternary complexes at atomic resolution. We present results demonstrating the efficiency, accuracy, and reliability of our approach to predict ternary structure ensembles using the bromodomain of SMARCA2 (SMARCA2 BD ) with the E3 ligase VHL as the system of interest. The simulations reproduce X-ray crystal structures – including prospective simulations validated on a new structure that we determined in this work (PDB ID: 7S4E) – with root mean square deviations (RMSD) of 1.1 to 1.6 Å. The simulations also reveal a structural ensemble of low-energy conformations of the ternary complex within a broad energy basin. To further characterize the structural ensemble, we used snapshots from the aforementioned simulations as seeds for Hamiltonian replica exchange molecular dynamics (HREMD) simulations, and then perform 7.1 milliseconds of aggregate simulation time using Folding@home. The resulting free energy surface identifies the crystal structure conformation within a broad low-energy basin and the dynamic ensemble is consistent with solution-phase biophysical experimental data (HDX-MS and small-angle x-ray scattering, SAXS). Finally, we graft structures from the ternary complexes onto the full CRL and perform enhanced sampling simulations, where we find that differences in degradation efficiency can be explained by the proximity distribution of lysine residues on the POI relative to the E2-loaded ubiquitin. Several of the top predicted ubiquitinated lysine residues are validated prospectively through a ubiquitin mapping proteomics experiment.

The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus?host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B?NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro. A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco's plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus?host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.

Caspases are cysteine-aspartic proteases involved in the regulation of programmed cell death (apoptosis) and a number of other biological processes. Despite overall similarities in structure and active-site composition, caspases show striking selectivity for particular protein substrates. Exosites are emerging as one of the mechanisms by which caspases can recruit, engage, and orient these substrates for proper hydrolysis. Following computational analyses and database searches for candidate exosites, we utilized site-directed mutagenesis to identify a new exosite in caspase-6 at the hinge between the disordered N-terminal domain (NTD), residues 23-45, and core of the caspase-6 structure. We observed that substitutions of the tri-arginine patch Arg-42-Arg-44 or the R44K cancer-associated mutation in caspase-6 markedly alter its rates of protein substrate hydrolysis. Notably, turnover of protein substrates but not of short peptide substrates was affected by these exosite alterations, underscoring the importance of this region for protein substrate recruitment. Hydrogen-deuterium exchange MS-mediated interrogation of the intrinsic dynamics of these enzymes suggested the presence of a substrate-binding platform encompassed by the NTD and the 240's region (containing residues 236-246), which serves as a general exosite for caspase-6-specific substrate recruitment. In summary, we have identified an exosite on caspase-6 that is critical for protein substrate recognition and turnover and therefore highly relevant for diseases such as cancer in which caspase-6-mediated apoptosis is often disrupted, and in neurodegeneration in which caspase-6 plays a central role.

Zinc is emerging as a widely used and important biological regulatory signal. Cellular zinc levels are tightly regulated by a complex array of zinc importers and exporters to control processes such as apoptotic cell death. While caspase inhibition by zinc has been reported previously, the reported inhibition constants were too weak to suggest a critical biological role for zinc-mediated inhibition. In this work, we have adopted a method of assessing available zinc. This allowed assessment of accurate inhibition constants for apoptotic caspases, caspase-3, -6, -7, and -8. Each of these caspases are inhibited by zinc at intracellular levels but with widely differing inhibition constants and different zinc binding stoichiometries. Caspase-3, -6, and -8 appear to be constitutively inhibited by typical zinc levels, and this inhibition must be lifted to allow activation. The inhibition constant for caspase-7 (76 nM) is much weaker than for the other apoptotic caspases (2.6-6.9 nM) suggesting that caspase-7 is not inactivated by normal zinc concentrations but can be inhibited under conditions of zinc stress. Caspase-3, -7, and -8 were found to bind three, one, and two zincs, respectively. In each of these caspases, zinc was present in the active site, in contrast to caspase-6, which binds one zinc allosterically. The most notable new mechanism to emerge from this work is for zinc-mediated inhibition of caspase-8. Zinc binds caspase-8 directly at the active site and at a second site. Zinc binding inhibits formation of the caspase-8 dimer, the activated form of the enzyme. Together these findings suggest that zinc plays a critical role in regulation of apoptosis by direct inactivation of caspases, in a manner that is unique for each caspase.

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. Here, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7 was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. This approach to specificity reprogramming should also be generalizable across a wide range of proteases.

Exosites for substrate binding are promising for development of inhibitors and activators with novel mechanisms of actions. Despite their potential, to date there is no systematic way to identify exosites on enzymes. We have developed a method for identifying exosites, and applied it to an important class of proteases, the caspases. To identify exosites, we built a version of caspase‐7 that maintained all exosites, but that has the active‐site specificity of caspase‐6. This new caspase allows us to categorize substrates as being active‐site or exosite driven. Simple substitution of the critical active‐site residues from caspase‐6 dramatically diminished caspase‐7 activity, but did not alter specificity, indicating that a more holistic evolutionary procedure would be needed to achieved the desired hybrid caspase. Using our GFP‐based reporter of caspase activity (CA‐GFP), we implemented a flow‐cytometry based directed evolution screen for caspase‐7 variants with caspase‐6 cleavage patterns. The caspase‐7 variants with the most strongly altered specificity displayed kinetic values similar to wild‐type caspase‐6 and mimicked caspase‐6 cleavage patterns with protein substrates. Our seven crystal structures of evolved caspases bound to either DEVD or VEID substrates allowed us to observe how the new residues in the active site contributed to the change in specificity by altering the conformation of a critical active site loop. Using N‐terminomics on the entire human proteome, we observed that the substrate specificity of the evolved caspase was superimposible with caspase‐6 and unlike the parent scaffold, caspase‐7, suggesting that our directed evolution was successful. This allowed us to identify Lamin A/C as an exosite dependent substrate. Ultimately we hope that this approach will identify exosites for recognition of just a subset of key regulatory or disease‐causing substrates, which will lead to more specific small‐molecule modulators of caspase function. Support or Funding Information This work was supported by the National Institutes of Health (GM80532) to JAH and (GM081051) to JAW.

Caspases are among the most specific of proteases, making them ideal targets for engineering new specificity and developing new protease‐based biotherapeutics. However, conventional high‐throughput methods for protein evolution are not amenable to caspases as they are multi‐chain, and intracellular. Increasing the complexity, active sites of caspases are highly flexible to allow for accommodation of various substrates, making a rational design approach difficult To overcome these challenges, we developed a method for evolving function in cytosolic proteases, based on our novel genetically‐encoded caspase activatable‐GFP reporter (CA‐GFP), which is activated from a dark to a fluorescent state by proteolysis.Our first application of this technology, we engineered caspase‐7, which cleaves the amino acid sequence DEVD to recognize and cleave the new sequence VEID, recognized by caspase‐6. Saturation mutagenesis at key residues within the active site of the enzyme allowed us to sort for variants with altered specificity by flow cytometry. Variants able to cleave the target sequence were purified, then characterized using traditional Michaelis‐Menten kinetics, protein substrate assays and x‐ray crystallography. We identified several variants that displayed kinetic activity and cleavage patterns similar to the wild type caspase‐6. X‐ray crystal structures of the evolved variants bound to the casp‐6 and ‐7 cognate substrates VEID, and DEVD reveal how the mutations affect the activity of the enzyme, which could not be rationally predicted, emphasizing the strength of this approach. Utilizing this method we are able to reengineer cytosolic proteases to cleave novel targets and pave the way for biotherapeutic applications.This work is supported in part by NIH GM080532.