Biology subjects

Wilde, K. L.

Publications and source records attributed to Wilde, K. L..

3 recordsLinked to original sources

bioRxiv2025

Dynamic allostery drives acetyl-CoA-mediated activation of Mycobacterium tuberculosis isocitrate lyase 2

Mycobacterium tuberculosis isocitrate lyase 2 (ICL2) is an allosterically regulated enzyme that enables the bacterium to survive on non-glycolytic substrates during infection. Previous studies showed that ICL2 is allosterically regulated by acetyl-CoA and its analogues but the molecular mechanism underpinning this regulation is unknown. Here, we use protein NMR, crystallography, molecular dynamics, and mutagenesis studies to show that two unique structural features of ICL2, its C-terminal domain and a unique helical substructure on its N-terminal catalytic domain, play important roles in the enzymes allostery. In particular, we found that the binding of acetyl-CoA promotes the dimerisation of the C-terminal domain and disrupts its interactions with the unique helical substructure on the N-terminal domain. This leads to conformational changes in the ICL2 enzyme that induces activation. Taken together, our findings reveal, for the first time, how the binding of acetyl-CoA, which is not an ICL2 substrate, induces ICL2 activation. By extension, the work also identifies a novel allosteric mechanism controlling M. tuberculosis metabolism that is amenable to therapeutic manipulation. Significance StatementMycobacterium tuberculosis isocitrate lyase 2 (ICL2) was previously shown to be activated by acetyl-CoA and propionyl-CoA - two central metabolites generated by the metabolism of sugars and fatty acids. However, it is not known how the binding of these metabolites leads to the activation of ICL2. Together with its isoform ICL1, ICL2 has been shown to be essential for the survival and pathogenesis of the bacterium. Understanding how this regulation occurs can help design novel treatments to target this protein and eradicate these bacteria, which cause the most deaths worldwide due to a single bacterial agent. This system also presents a fascinating model to examine allostery in proteins, with the techniques illustrated in this paper being applicable to other allosteric proteins.

biochemistry↗

bioRxiv2025

Structural Studies of an Anti-necroptosis Viral:Human Functional Hetero-amyloid M45:RIPK3 using SSNMR

The formation of RIP-homotypic interaction motif (RHIM)-based heteromeric amyloid assemblies between effector proteins such as RIPK1, ZBP1, or TRIF and the kinase RIPK3 serve as regulating signals for the necroptosis process, a key element of innate immune defense. Murine cytomegalovirus (MCMV) expresses the M45-encoded viral inhibitor of RIP activation (vIRA) which inhibits necroptosis in a RHIM-dependent manner. A pivotal question is how viral M45 forms hetero-amyloids with RIPK3 to effectively create an inhibitory assembly. We report a novel high-resolution structure of the M45:RIPK3 complex where M45 and RIPK3 alternately stack in an amyloid-state structure. Mutagenesis of the residues flanking the IQIG tetrad in M45 results in specific impacts on co-assembly with RIPK3, indicating an extended interface in the heteromeric fibrils. Other key interactions support the formation of stable viral:host fibrils. The M45: RIPK3 hetero-amyloid is likely to act as an anti-necroptotic signal by competing with formation of other pro-necroptotic species and introducing a barrier to RIPK3 autophosphorylation. Significance StatementThis study investigates the structural biology of the necroptotic pathway, an understudied programmed cell death mechanism that plays a crucial role in innate immunity and has implications for infectious diseases, cell cycle regulation, and cancer. We present the high-resolution structure of a cross-species hetero-amyloid in which M45, a murine cytomegalovirus (MCMV) protein, co-assembles with human RIPK3 to inhibit necroptosis by competing with pro-necroptotic amyloids. Using solid-state NMR, cryo-EM, mutagenesis, and biophysical analyses, we uncover a novel structural paradigm for cross-species hetero-amyloids, shedding light on viral strategies to manipulate host immunity and protein interactions.

biophysics↗

bioRxiv2021

Membrane permeabilization is mediated by distinct epitopes in mouse and human orthologs of the necroptosis effector, MLKL

Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKLs N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the 3 helix, W108 in the 4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the 1 and 2 helices. In further contrast to human and plant MLKL orthologs, in which the 3-4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.

cell biology↗