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Updated: Jan 8, 2026
X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Published on: May 13, 2020
Structural characterisation of the fungal Pmt4 homodimer
Melanie A McDowell1,2, Klemens Wild3, Francesco Fiorentino4,5
1Heidelberg University Biochemistry Centre (BZH), Heidelberg, Germany. melanie.mcdowell@biophys.mpg.de.
Abstract:
Protein O-mannosyltransferases (PMTs) are conserved endoplasmic reticulum membrane-embedded enzymes responsible for the transfer of mannose from dolichol phosphate-mannose (Dol-P-Man) to serine/threonine-rich protein substrates or unfolded proteins. PMTs from three subfamilies form obligate dimers with different substrate specificities and require the concerted action of their transmembrane domains (TMDs) and a luminal MIR domain for catalysis. Here, we present structures, native mass spectrometry, and structure-based mutagenesis of the fungal Pmt4 homodimer. The core fold of the TMDs and MIR domain is conserved with the Pmt1-Pmt2 heterodimer, indicating a shared catalytic mechanism. Distinct from Pmt4, the MIR domain interacts in cis with the TMDs of the same subunit and has a β-hairpin insertion required for O-mannosylation of substrates. We further identify a cytosolic binding site for substrate Dol-P-Man within the Pmt4 TMDs, which is conserved amongst PMTs and important for in vivo activity. Thus, we provide a framework to understand the substrate specificity and regulation of the Pmt4 homodimer.
Insights
Protein O-mannosyltransferases (PMTs) are ER enzymes that transfer mannose to proteins. This study reveals the Pmt4 homodimer structure, highlighting conserved mechanisms and unique features for substrate binding and O-mannosylation.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Protein O-mannosyltransferases (PMTs) are crucial endoplasmic reticulum enzymes.
- PMTs catalyze mannose transfer from dolichol phosphate-mannose (Dol-P-Man) to proteins.
- PMTs function as heterodimers or homodimers, with varying substrate specificities.
Purpose of the Study:
- To elucidate the structure and function of the fungal Pmt4 homodimer.
- To understand the conserved and distinct catalytic mechanisms of PMTs.
- To identify key structural elements involved in substrate binding and O-mannosylation.
Main Methods:
- X-ray crystallography for structural determination.
- Native mass spectrometry for analyzing protein complexes.
- Structure-based mutagenesis to assess functional importance.
Main Results:
- The Pmt4 homodimer shares a conserved core fold with the Pmt1-Pmt2 heterodimer.
- The Pmt4 MIR domain interacts in cis with its transmembrane domains (TMDs).
- A novel cytosolic binding site for Dol-P-Man was identified in the Pmt4 TMDs.
Conclusions:
- The study provides a structural framework for Pmt4 homodimer function.
- Conserved and unique features explain substrate specificity and regulation in PMTs.
- The identified cytosolic binding site is critical for PMT activity in vivo.
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