Rewiring V-type and K-type enzyme allostery through subunit interface mutations
Abstract
Allosteric regulation in the heterodimeric enzyme IGPS depends on long-range communication between the effector-binding HisF subunit and the catalytic HisH subunit. This signaling occurs through a densely packed interdomain interface enriched in conserved noncovalent contacts. Here, we use targeted interface mutations to determine how specific interfacial contacts tune the structural and dynamic features that govern allosteric control. The hK181A variant, which disrupts a critical salt bridge with fD98, converts IGPS into a constitutively more active enzyme, increasing basal glutaminase activity and substrate affinity. By contrast, hR18A, which disrupts a secondary salt bridge with fE71, weakens effector-induced activation, revealing functional asymmetry among interfacial interactions. NMR chemical shift perturbation and CPMG relaxation dispersion experiments show that hK181A remodels millisecond-timescale dynamics throughout HisF, consistent with molecular dynamics simulations indicating enhanced sampling of catalytically competent conformations. Network traffic analysis of correlated communication pathways, combined with energetic analysis, further shows that enthalpic and entropic contributions are redistributed to rewire long-range allosteric signaling. Together, these results identify specific interfacial residues as molecular gates that shape the conformational ensemble accessible to IGPS and show how interface reengineering can be used to rationally reprogram allosteric output. Significance Statement Enzymes often work like molecular switches: binding at one site can change activity at another distant site. How to predict or redesign this communication remains a major challenge. Using imidazole glycerol phosphate synthase (IGPS), we show that changing single amino acids at the interface between its two protein subunits can alter how the enzyme responds to regulation. One substitution shifts IGPS toward a response that changes both catalytic rate and substrate binding, whereas another weakens activation by disrupting communication across the interface. These findings identify interfacial residues that act as control points in an allosteric network and suggest a practical strategy for engineering enzymes with customized regulatory behavior.
Related
- Cryo-tides: synthetic peptides for cryo-EM grid preparation of multi-subunit protein complexes sensitive to the air-water interface
- The Intersection of Allostery and Induced Proximity through the E3 Ligase Cereblon
- Chromatin-modifying Complex Component Nurf55/p55 Associates with Histones H3 and H4 and Polycomb Repressive Complex 2 Subunit Su(z)12 through Partially Overlapping Binding Sites
- Ebselen restores mitochondrial function through metabolic rewiring in Barth syndrome and related mitochondrial diseases
- Disease mutations in the PWWP domain of DNMT3A affect chromatin recruitment through multiple mechanisms
BibTeX
@misc{chaudhuri2026rewiring,
title = {{Rewiring V-type and K-type enzyme allostery through subunit interface mutations}},
author = {Apala Chaudhuri and Federica Maschietto and Olivia Enny and Victor Batista and J. Patrick Loria},
howpublished = {Bioarxiv},
year = {2026},
month = jun,
doi = {10.64898/2026.05.29.728626},
url = {https://hal.science/hal-05850441v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Rewiring V-type and K-type enzyme allostery through subunit interface mutations
AU - Apala Chaudhuri
AU - Federica Maschietto
AU - Olivia Enny
AU - Victor Batista
AU - J. Patrick Loria
PY - 2026
DA - 2026/06/02
PB - Bioarxiv
DO - 10.64898/2026.05.29.728626
UR - https://hal.science/hal-05850441v1
AB - Allosteric regulation in the heterodimeric enzyme IGPS depends on long-range communication between the effector-binding HisF subunit and the catalytic HisH subunit. This signaling occurs through a densely packed interdomain interface enriched in conserved noncovalent contacts. Here, we use targeted interface mutations to determine how specific interfacial contacts tune the structural and dynamic features that govern allosteric control. The hK181A variant, which disrupts a critical salt bridge with fD98, converts IGPS into a constitutively more active enzyme, increasing basal glutaminase activity and substrate affinity. By contrast, hR18A, which disrupts a secondary salt bridge with fE71, weakens effector-induced activation, revealing functional asymmetry among interfacial interactions. NMR chemical shift perturbation and CPMG relaxation dispersion experiments show that hK181A remodels millisecond-timescale dynamics throughout HisF, consistent with molecular dynamics simulations indicating enhanced sampling of catalytically competent conformations. Network traffic analysis of correlated communication pathways, combined with energetic analysis, further shows that enthalpic and entropic contributions are redistributed to rewire long-range allosteric signaling. Together, these results identify specific interfacial residues as molecular gates that shape the conformational ensemble accessible to IGPS and show how interface reengineering can be used to rationally reprogram allosteric output. Significance Statement Enzymes often work like molecular switches: binding at one site can change activity at another distant site. How to predict or redesign this communication remains a major challenge. Using imidazole glycerol phosphate synthase (IGPS), we show that changing single amino acids at the interface between its two protein subunits can alter how the enzyme responds to regulation. One substitution shifts IGPS toward a response that changes both catalytic rate and substrate binding, whereas another weakens activation by disrupting communication across the interface. These findings identify interfacial residues that act as control points in an allosteric network and suggest a practical strategy for engineering enzymes with customized regulatory behavior.
ER -