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<p>Human <i>cis</i>-prenyltransferase (h<i>cis</i>-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 h<i>cis</i>-PT. By solving the apo structure of h<i>cis</i>-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 NgBR<sup>S249</sup> 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 <i>cis</i>-prenyltransferases and suggests avenues for therapeutic targeting of h<i>cis</i>-PT-related disorders.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月28日

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  • Moshe Giladi,
  • Shiri Kredi,
  • Carlo Guardiani,
  • Lyr Aviram,
  • Pavla Vaňková,
  • Yarin Gaizinger,
  • Petr Man,
  • Alberto Giacomello,
  • Yoni Haitin

摘要

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 avenues for therapeutic targeting of hcis-PT-related disorders.