Biology subjects

Gross, M. L.

Publications and source records attributed to Gross, M. L..

6 recordsLinked to original sources

bioRxiv2026

Protonation- and substrate-regulated dimer opening couples brain-type creatine kinase to vesicular and actin-remodeling membranes

Brain-type creatine kinase (CK-BB) buffers local ATP demand through reversible phosphotransfer between ATP and phosphocreatine, yet how this soluble metabolic enzyme engages membrane compartments is unknown. Here, we combine fluorescence microscopy, DEER spectroscopy, hydrogen-deuterium exchange and native mass spectrometry, DEER- and AlphaFold-guided modeling, and long-timescale molecular dynamics to define the pH- and substrate-regulated conformational landscape governing CK-BB membrane association. Acidification promotes curvature-sensitive membrane binding and redistributes endogenous and recombinant CK-BB from diffuse cytosolic pools to punctate vesicular structures and membrane ruffles. Substrates independently promote curvature-sensitive association at neutral pH. DEER and modeling reveal an asymmetric dimer in which the convex surface remains restrained, whereas the concave catalytic-regulatory surface samples pH- and substrate-dependent intermediates. We identify progressive dimer opening as a novel regulatory mechanism whereby acidification and substrate binding increase dynamics across the convex surface and N-terminal dimer interface, generating membrane-competent conformations that facilitate curvature sensing and membrane association. Substrate binding buffers acid-induced deprotection while preserving dynamics near the His191/Ser199 regulatory interface. These findings establish CK-BB as a previously unrecognized curvature-sensitive metabolic enzyme and define dimer dynamics as a molecular switch coupling protonation and substrate occupancy to curved-membrane recognition and localized ATP regeneration, with potential relevance to neurodegeneration and cellular stress.

biophysics↗

bioRxiv2025

A SWI/SNF-specific Ig-like domain, SWIFT, is a transcription factor binding platform

Mammalian SWI/SNF (BAF) chromatin remodeling complexes modulate DNA accessibility and gene expression, however, their genomic targeting mechanisms remain incompletely understood. Here, we identify SWIFT (SWI/SNF Ig-Fold for Transcription Factor Interactions), a conserved, broad transcription factor (TF) binding platform on the SMARCD subunits. SWIFT is necessary and sufficient for direct engagement with the transactivation domain of PU.1, a single mutation in which disrupts PU.1-mSWI/SNF binding, impairs complex targeting, and attenuates oncogenic transcription and proliferation in PU.1-dependent cancer cells. Dominant expression of the SWIFT domain in isolation sequesters TFs from mSWI/SNF and poisons TF-addicted cancer cells. Finally, TFs across diverse families interact with SMARCD paralog-specific SWIFT domains. These results define a major mechanism of cell type- and disease-specific mSWI/SNF chromatin targeting and inform approaches toward therapeutic modulation.

molecular biology↗

bioRxiv2022

Small Molecule Modulation of the Archetypal UbiB protein COQ8

Small molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize inhibitor and activator molecules for the archetypal UbiB member, COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays, and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Second, using 1H-13C HMQC NMR and hydrogen-deuterium exchange mass spectrometry, we reveal that the CoQ precursor mimetic, 2-propylphenol (2-PP), modulates the quintessential UbiB KxGQ domain to increase COQ8A nucleotide affinity and ATPase activity. Our newfound chemical tools promise to lend new mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.

biochemistry↗

bioRxiv2020

Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain

Nucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. We also map the RNA binding interface using hydrogen-deuterium exchange mass spectrometry. Finally, we find that the N protein C-terminal domain is the most immunogenic by sensitivity, based upon antibody binding to COVID-19 patient samples from the US and Hong Kong. Together, these findings uncover domain-specific insights into the significance of SARS-CoV-2 N and highlight the diagnostic value of using N domains as highly specific and sensitive markers of COVID-19.

biochemistry↗

bioRxiv2020

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs. In these interactions the largely alpha-helical carboxy-terminal domain of the EBOV VP30 engages with the motif such that the prolines adopt non-canonical orientations, as compared to other proline-rich motifs. Affinity tag-purification mass spectrometry identified additional PPxPxY-containing host proteins, including hnRNP L, hnRNPUL1 and PEG10, as VP30 interactors. Of these, hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV replication, whereas hnRNPUL1 enhances. Further, double knockdown studies support additive effects of RBBP6 and hnRNP L. Binding studies demonstrate variable capacity of PPxPxY motifs to bind VP30 and the extended motif PxPPPPxY is demonstrated to confer optimal binding and to inhibit RNA synthesis, with the fifth proline and the tyrosine being most critical. Competition binding and hydrogen-deuterium exchange studies demonstrate that each protein binds a similar interface on VP30 and impacts VP30 phosphorylation. VP30 therefore represents a novel proline recognition domain that allows multiple host proteins to target a single viral protein-protein interface to modulate viral transcription.

microbiology↗

bioRxiv2019

A Novel Antenna Protein Complex in the Life Cycle of Cyanobacterial Photosystem II

In oxygenic photosynthetic organisms, photosystem II (PSII) is a unique membrane protein complex that catalyzes light-driven oxidation of water. PSII undergoes frequent damage due to its demanding photochemistry. However, many facets of its repair and reassembly following photodamage remain unknown. We have discovered a novel PSII subcomplex that lacks five key PSII core reaction center polypeptides: D1, D2, PsbE, PsbF, and PsbI. This pigment-protein complex does contain the PSII core antenna proteins CP47 and CP43, as well as most of their associated low-molecular-mass subunits, and the assembly factor Psb27. Immunoblotting analysis, multiple mass spectrometry techniques, and ultrafast spectroscopic results supported the absence of a functional reaction center in this chlorophyll-protein complex. We therefore refer to it as the no reaction center complex (NRC). Additionally, genetic deletion of PsbO on the PSII lumenal side resulted in an increased NRC population, indicative of a faulty PSII repair scheme at the cellular level. Analytical ultracentrifugation studies and clear native acrylamide gel analysis showed that the NRC complex is a stable pigment-protein complex and not a mixture of free CP47 and CP43 proteins. Our finding challenges the current model of the PSII repair cycle and implies an alternative PSII repair strategy. We propose that formation of this pigment-protein complex maximizes PSII repair economy by preserving an intact PSII core antenna shell in a single complex that is available for PSII reassembly, thus minimizing the risk of randomly diluting multiple recycling components in the thylakoid membrane following a photodamage event at the RC.\n\nSignificance statementPhotosystem II (PSII) converts sunlight into chemical energy, powering nearly all life on Earth. The efficiency of this process is maximized under various environmental conditions by a frequent repair and reassembly cycle that follows inevitable PSII damage even during normal oxygenic photosynthesis. We have isolated a novel pigment protein PSII subcomplex in which, surprisingly, the reaction center (RC) components of PSII are absent. Formation of this stable chlorophyll-protein complex suggests a protective mechanism whereby longer-lived PSII subunits are unplugged from the damaged RC to prevent harmful, aberrant photochemistry during RC repair. This finding provides intriguing new insight into how PSII is assembled and rebuilt to optimize its performance to optimally catalyze one of the most challenging reactions in biology.

plant biology↗