Functional implications of MIR domains in protein O-mannosylation.

Antonella Chiapparino,Antonija Grbavac,Gunter Stier,Sabine Strahl,Klemens Wild,Krishna Saxena,Yvonne Hackmann,Hendrik Ra Jonker,Ewa Zatorska,Harald Schwalbe,Irmgard Sinning,Sofia Mortensen,Andrea Schott

doi:10.7554/elife.61189

Antonella Chiapparino, Antonija Grbavac + Show 11 more

Open Access

https://doi.org/10.7554/elife.61189

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Journal: eLife	Publication Date: Dec 24, 2020
Citations: 3	License type: CC BY 4.0

Affiliation: Heidelberg University, Goethe University Frankfurt

Abstract

Protein O-mannosyltransferases (PMTs) represent a conserved family of multispanning endoplasmic reticulum membrane proteins involved in glycosylation of S/T-rich protein substrates and unfolded proteins. PMTs work as dimers and contain a luminal MIR domain with a β-trefoil fold, which is susceptive for missense mutations causing α-dystroglycanopathies in humans. Here, we analyze PMT-MIR domains by an integrated structural biology approach using X-ray crystallography and NMR spectroscopy and evaluate their role in PMT function in vivo. We determine Pmt2- and Pmt3-MIR domain structures and identify two conserved mannose-binding sites, which are consistent with general β-trefoil carbohydrate-binding sites (α, β), and also a unique PMT2-subfamily exposed FKR motif. We show that conserved residues in site α influence enzyme processivity of the Pmt1-Pmt2 heterodimer in vivo. Integration of the data into the context of a Pmt1-Pmt2 structure and comparison with homologous β-trefoil - carbohydrate complexes allows for a functional description of MIR domains in protein O-mannosylation.

Highlights

Protein O-mannosyltransferases (PMTs) of the PMT family are multispanning membrane glycosyltransferases (GT family 39; http://www.cazy.org) of the GT-C fold (Albuquerque-Wendt et al, 2019; Bai et al, 2019) that catalyze the transfer of mannose from dolichol monophosphate-activated mannose (Dol-P-Man) to the hydroxyl group of serine and threonine residues of proteins in the endoplasmic reticulum (ER)
While MIR domains of the PMT1 family proved to be unstable when analyzed by nano differential scanning fluorimetry and failed to crystallize, PMT2 family MIR domains were more thermostable and readily formed crystals
X-ray structures were determined by molecular replacement using the MIR domain of the stromal-derived factor 2 (SDF2) from Arabidopsis thaliana previously solved in our laboratory (Radzimanowski et al, 2010; Schott et al, 2010; Figure 1B,C and Table 1)

Summary

Introduction

Protein O-mannosyltransferases (PMTs) of the PMT family are multispanning membrane glycosyltransferases (GT family 39; http://www.cazy.org) of the GT-C fold (Albuquerque-Wendt et al, 2019; Bai et al, 2019) that catalyze the transfer of mannose from dolichol monophosphate-activated mannose (Dol-P-Man) to the hydroxyl group of serine and threonine residues of proteins in the endoplasmic reticulum (ER) (reviewed in Neubert and Strahl, 2016). O-Mannosylation of PMT bona fide substrates occurs mainly in S/T-rich protein segments (Larsen et al, 2017; Neubert et al, 2016), whereas in ER protein quality control (referred to as unfolded protein O-mannosylation; UPOM) PMTs target isolated serines and threonines of un- or misfolded proteins (reviewed in Xu and Ng, 2015). Structural and functional studies on PMT proteins carried out to date do not answer the question of whether PMT-MIR domains bind to the mannose of the Dol-P-Man substrate or the O-Man peptide product, and if or how they contribute to O-mannosylation of both bona fide substrates and UPOM target proteins. The importance of the MIR domain for O-mannosylation and disease are discussed in the context of the structure of the Pmt1-Pmt holoenzyme and related glycosyltransferases

Results

Discussion

Materials and methods

Funding Funder German Research Foundation