Predicting the structural basis of targeted protein degradation by integrating molecular dynamics simulations with structural mass spectrometry.

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.

A fluorescence-activatable reporter of flavivirus NS2B-NS3 protease activity enables live imaging of infection in single cells and viral plaques.

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.

Tri-arginine exosite patch of caspase-6 recruits substrates for hydrolysis.

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.

Multiple Mechanisms of Zinc-Mediated Inhibition for the Apoptotic Caspases-3, -6, -7, and -8.

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.

Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition.

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.