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Structural mechanisms of allosteric regulation in the human cis-prenyltransferase complex

ABSTRACT: Human cis-prenyltransferase (hcis-PT) synthesizes long-chain isoprenoids essential for N-linked protein glycosylation. This heteromeric complex comprises the catalytic subunit DHDDS and the regulatory Nogo-B receptor (NgBR). Although NgBR dramatically enhances DHDDS activity, the molecular basis for this allosteric regulation remains unclear. Here, we combined crystallography, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics simulations, and network analysis to uncover the structural dynamics and communication pathways within hcis-PT. By solving the apo structure of hcis-PT, we reveal only a localized flexibility at the active site and the NgBR C-terminus. However, HDX-MS demonstrated widespread substrate-induced stabilization, particularly at the NgBR βD–βE loop, highlighting it as an allosteric hub. Functional mutagenesis scanning identified NgBRS249 as critical for enzymatic activity, independent of structural perturbations. Network analysis of MD simulations pinpointed this residue as a central node in inter-subunit communication, with perturbations disrupting downstream allosteric pathways, altering enzymatic activity. Our findings reveal a dynamic regulatory network centered at the inter-subunit interface, wherein specific NgBR residues modulate DHDDS activity through allosteric signaling. This work elucidates a conserved mechanism of subunit coordination in long-chain cis-prenyltransferases and suggests novel avenues for therapeutic targeting of hcis-PT-related disorders.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Petr Man

LAB HEAD: Petr Man

PROVIDER: PXD063722 | Pride | 2025-10-20

REPOSITORIES: Pride

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			Action	DRS
	DHDDS-construct.fasta	Fasta
	DHDDS.fasta	Fasta
	HDX-results-DHDDS.xlsx	Xlsx
	HDX-results-NgBR.xlsx	Xlsx
	NBGR.fasta	Fasta

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