Membrane association of monotopic phosphoglycosyl transferase underpins function

Ray, Leah; Das, Debasis; Entova, Sonya; Lukose, Vinita; Lynch, A.J.; Imperiali, Barbara; Allen, Karen N.

doi:10.1038/s41589-018-0054-z

Cited by 43 publications

(91 citation statements)

References 35 publications

(57 reference statements)

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“…The crystal structure of PglC shows that the N-terminal hydrophobic domain forms a reentrant membrane helix (RMH) that anchors the fold in the membrane ( Ray et al, 2018 ). However, as the reported structure was generated using detergent-solubilized PglC in an aqueous environment, we applied molecular dynamics (MD) simulations to investigate whether the structure would be stable in a more native membrane-like environment.…”

Section: Resultsmentioning

confidence: 99%

“…In the reported structure, the N-terminal domain of PglC forms a reentrant helix-break-helix structure, termed the reentrant membrane helix (RMH), such that both the N-terminus and the globular domain, which includes the active site, are on the cytoplasmic face of the inner membrane ( Figure 1A ). A reentrant topology was further confirmed in vivo using a substituted cysteine accessibility method (SCAM) ( Nasie et al, 2013 ; Ray et al, 2018 ). As such, the same reentrant topology is confirmed in both PglC and the corresponding domain of an elaborated WbaP homolog, suggesting that the topology and mechanism of membrane association enforced by the RMH is a conserved feature of the PGTs in this extensive superfamily.…”

Section: Introductionmentioning

confidence: 94%

“…Recently, we reported the X-ray structure of PglC from Campylobacter concisus to a resolution of 2.7 Å (PDB 5W7L) ( Ray et al, 2018 ). The structural analysis complements previous biochemical studies on PglC from C. jejuni ; the homologs share 72% sequence identity.…”

Section: Introductionmentioning

confidence: 99%

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Insights into the key determinants of membrane protein topology enable the identification of new monotopic folds

Entova

Billod

Swiecicki

et al. 2018

eLife

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Monotopic membrane proteins integrate into the lipid bilayer via reentrant hydrophobic domains that enter and exit on a single face of the membrane. Whereas many membrane-spanning proteins have been structurally characterized and transmembrane topologies can be predicted computationally, relatively little is known about the determinants of membrane topology in monotopic proteins. Recently, we reported the X-ray structure determination of PglC, a full-length monotopic membrane protein with phosphoglycosyl transferase (PGT) activity. The definition of this unique structure has prompted in vivo, biochemical, and computational analyses to understand and define key motifs that contribute to the membrane topology and to provide insight into the dynamics of the enzyme in a lipid bilayer environment. Using the new information gained from studies on the PGT superfamily we demonstrate that two motifs exemplify principles of topology determination that can be applied to the identification of reentrant domains among diverse monotopic proteins of interest.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%

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Insights into the key determinants of membrane protein topology enable the identification of new monotopic folds

Entova

Billod

Swiecicki

et al. 2018

eLife

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“…Unlike traditional transmembrane proteins, monotopic membrane proteins contain structural elements that enter and exit on a single face of the membrane as opposed to completely spanning the membrane bilayer. These proteins have been particularly recalcitrant for structural studies, as there are only a few with available structures (10,34,35). The vast majority of the monotopic membrane proteins with known structures are enzymes that share homology with soluble proteins that catalyze similar reactions.…”

Section: Caveolin As a Model For Understanding Monotopic Membrane Promentioning

confidence: 99%

Structure and assembly of CAV1 8S complexes revealed by single particle electron microscopy

Han

Porta

Hanks

et al. 2020

Preprint

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Short title: Single particle analysis of caveolin-1 complexesOne sentence summary. Single particle electron microscopy reveals the overall structural organization of oligomeric complexes of an essential structural component of caveolae, the monotopic membrane protein caveolin-1.Abbreviations: Caveolin (Cav), electron microscopy (EM), heterologous caveolae (h-caveolae), wild type (WT), Maltose binding protein (MBP), congenital generalized lipodystrophy (CGL), pulmonary arterial hypertension (PAH), Fast protein liquid chromatography (FPLC). AbstractHighly stable oligomeric complexes of the monotopic membrane protein caveolin serve as fundamental building blocks of caveolae. Current evidence suggests these complexes are disc shaped, but the details of their structural organization and how they assemble are poorly understood. Here, we address these questions using single particle electron microscopy of negatively stained recombinant 8S complexes of human Caveolin-1. We show that 8S complexes are toroidal structures ~15 nm in diameter that consist of an outer ring, an inner ring, and central protruding stalk. Moreover, we map the position of the N-and C-termini and determine their role in complex assembly, and visualize the 8S complexes in heterologous caveolae. Our findings provide critical insights into the structural features of 8S complexes and allow us to propose a new model for how these highly stable membrane-embedded complexes are generated.

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“…The results represent measurements from eight independent experiments. of them are available (Monk et al, 2014;Ray et al, 2018). The concentrated PA2949 sample (3.5 mg ml À1 ) was centrifuged (21 000g, 10 min) prior to performing crystallization setups.…”

Section: Figurementioning

confidence: 99%

Pseudomonas aeruginosaesterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization

et al. 2019

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The human membrane-bound α/β-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane-bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2-AG and esters of medium-length fatty acids. PA2949 in the presence of n-octyl β-D-glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified from Escherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space group I4122 and diffracted to a resolution of 2.54 Å. This study provides hints on the functional similarity of ABHD6-like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult-to-crystallize proteins.

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Membrane association of monotopic phosphoglycosyl transferase underpins function

Cited by 43 publications

References 35 publications

Insights into the key determinants of membrane protein topology enable the identification of new monotopic folds

Insights into the key determinants of membrane protein topology enable the identification of new monotopic folds

Structure and assembly of CAV1 8S complexes revealed by single particle electron microscopy

Pseudomonas aeruginosaesterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization

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