Erratum to: Charged residues next to transmembrane regions revisited: “Positive-inside rule” is complemented by the “negative inside depletion/outside enrichment rule”

Baker, James Alexander; Wong, Wing-Cheong; Eisenhaber, Birgit; Warwicker, Jim; Eisenhaber, Frank

doi:10.1186/s12915-017-0410-6

Cited by 7 publications

(6 citation statements)

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“…Further, we wish to emphasize that the TM regions in TMTCs are largely of the complex type (the only consistently simple TMs are TM7 in TMTC3 from various species (data not shown)) [ 66 , 67 ]; thus, their sequences contain evolutionary information beyond the generally not informative hydrophobic background (sprinkled-in polar residues, glycine and proline are typically rare in TMs [ 68 , 69 ]) useful for sequence comparison in homology searches [ 70 – 72 ].…”

Section: Resultsmentioning

confidence: 99%

Conserved sequence motifs in human TMTC1, TMTC2, TMTC3, and TMTC4, new O-mannosyltransferases from the GT-C/PMT clan, are rationalized as ligand binding sites

et al. 2021

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Background The human proteins TMTC1, TMTC2, TMTC3 and TMTC4 have been experimentally shown to be components of a new O-mannosylation pathway. Their own mannosyl-transferase activity has been suspected but their actual enzymatic potential has not been demonstrated yet. So far, sequence analysis of TMTCs has been compromised by evolutionary sequence divergence within their membrane-embedded N-terminal region, sequence inaccuracies in the protein databases and the difficulty to interpret the large functional variety of known homologous proteins (mostly sugar transferases and some with known 3D structure). Results Evolutionary conserved molecular function among TMTCs is only possible with conserved membrane topology within their membrane-embedded N-terminal regions leading to the placement of homologous long intermittent loops at the same membrane side. Using this criterion, we demonstrate that all TMTCs have 11 transmembrane regions. The sequence segment homologous to Pfam model DUF1736 is actually just a loop between TM7 and TM8 that is located in the ER lumen and that contains a small hydrophobic, but not membrane-embedded helix. Not only do the membrane-embedded N-terminal regions of TMTCs share a common fold and 3D structural similarity with subgroups of GT-C sugar transferases. The conservation of residues critical for catalysis, for binding of a divalent metal ion and of the phosphate group of a lipid-linked sugar moiety throughout enzymatically and structurally well-studied GT-Cs and sequences of TMTCs indicates that TMTCs are actually sugar-transferring enzymes. We present credible 3D structural models of all four TMTCs (derived from their closest known homologues 5ezm/5f15) and find observed conserved sequence motifs rationalized as binding sites for a metal ion and for a dolichyl-phosphate-mannose moiety. Conclusions With the results from both careful sequence analysis and structural modelling, we can conclusively say that the TMTCs are enzymatically active sugar transferases belonging to the GT-C/PMT superfamily. The DUF1736 segment, the loop between TM7 and TM8, is critical for catalysis and lipid-linked sugar moiety binding. Together with the available indirect experimental data, we conclude that the TMTCs are not only part of an O-mannosylation pathway in the endoplasmic reticulum of upper eukaryotes but, actually, they are the sought mannosyl-transferases.

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Section: Resultsmentioning

confidence: 99%

Conserved sequence motifs in human TMTC1, TMTC2, TMTC3, and TMTC4, new O-mannosyltransferases from the GT-C/PMT clan, are rationalized as ligand binding sites

et al. 2021

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“…The mutation K382E was found to be associated with hypertension (Dewey et al, 2016). Positively charged residues are more abundant in the cytoplasmic side of many membrane proteins (“positive-inside rule”) and the substitution of such residue with an opposite charge could potentially affect the proper insertion and orientation of the protein to the membrane (Baker et al, 2017; Sipos and von Heijne, 1993; von Heijne, 2006). Notably, the positively charged K382 is located far from the cation-binding site, facing the cytoplasm (Fig.…”

Section: Discussionmentioning

confidence: 99%

Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na⁺/H⁺ antiporter NHA2

Velázquez

Průša

Masrati

et al. 2022

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The human Na+/H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity and increased resistance to the HsNHA2-specific inhibitor phloretin that were not only highly active at an acidic pH of 4.0 (like the native antiporter), but also at neutral pH. We also experimentally confirmed the importance of a putative salt bridge between E215 and R432 for antiporter function and structural integrity. Truncations of the first 50 - 70 residues of the N-terminus doubled the transport activity of HsNHA2, whilst changes in the charge at positions E47, E56, K57, or K58 decreased the antiporter’s transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically autoinhibit its cation transport. Our data also show this in vivo approach to be useful for a rapid screening of SNP’s effect on HsNHA2 activity.

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“…39 Positively charged residues are more abundant in the cytoplasmic side of many membrane proteins ("positive-inside rule") and the substitution of such residue with an opposite charge could potentially affect the proper insertion and orientation of the protein to the membrane. [72][73][74] Notably, the positively charged K382 is located far from the cation-binding site, facing the cytoplasm (Figure 8d), its replacement with negatively charged glutamate did not change the plasmamembrane localization of the antiporter (Figure 5b), but it resulted in restricted substrate specificity for Li + alone and very low transport activity (ability to improve the LiCl tolerance of cells) (Figure 5a). On the other hand, the V240L mutation in the antiporter's core domain (Figure 8d,e) had only minor effect on HsNHA2's functionality (Figure 5), probably, due to its conservative nature.…”

Section: Discussionmentioning

confidence: 99%

“…The mutation K382E was found to be associated with hypertension (Dewey et al, 2016). Positively charged residues are more abundant in the cytoplasmic side of many membrane proteins ("positive-inside rule") and the substitution of such residue with an opposite charge could potentially affect the proper insertion and orientation of the protein to the membrane (Baker et al, 2017;Sipos and von Heijne, 1993;von Heijne, 2006). Notably, the positively charged K382 is located far from the cation-binding site, facing the cytoplasm (Fig.…”

Section: Mutations Of Proline 246 To G a S Or T Provided The Antiport...mentioning

confidence: 99%

Allosteric links between the hydrophilic N‐terminus and transmembrane core of human Na⁺/H⁺ antiporter NHA2

et al. 2022

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The human Na + /H + antiporter NHA2 (SLC9B2) transports Na + or Li + across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity and increased resistance to the HsNHA2-specific inhibitor phloretin that were not only highly active at an acidic pH of 4.0 (like the native antiporter), but also at neutral pH. We also experimentally confirmed the importance of a putative salt bridge between E215 and R432 for antiporter function and structural integrity. Truncations of the first 50 -70 residues of the Nterminus doubled the transport activity of HsNHA2, whilst changes in the charge at positions E47, E56, K57, or K58 decreased the antiporter's transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically autoinhibit its cation transport. Our data also show this in vivo approach to be useful for a rapid screening of SNP´s effect on HsNHA2 activity.

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Erratum to: Charged residues next to transmembrane regions revisited: “Positive-inside rule” is complemented by the “negative inside depletion/outside enrichment rule”

Cited by 7 publications

References 1 publication

Conserved sequence motifs in human TMTC1, TMTC2, TMTC3, and TMTC4, new O-mannosyltransferases from the GT-C/PMT clan, are rationalized as ligand binding sites

Conserved sequence motifs in human TMTC1, TMTC2, TMTC3, and TMTC4, new O-mannosyltransferases from the GT-C/PMT clan, are rationalized as ligand binding sites

Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na⁺/H⁺ antiporter NHA2

Allosteric links between the hydrophilic N‐terminus and transmembrane core of human Na⁺/H⁺ antiporter NHA2

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Erratum to: Charged residues next to transmembrane regions revisited: “Positive-inside rule” is complemented by the “negative inside depletion/outside enrichment rule”

Cited by 7 publications

References 1 publication

Conserved sequence motifs in human TMTC1, TMTC2, TMTC3, and TMTC4, new O-mannosyltransferases from the GT-C/PMT clan, are rationalized as ligand binding sites

Conserved sequence motifs in human TMTC1, TMTC2, TMTC3, and TMTC4, new O-mannosyltransferases from the GT-C/PMT clan, are rationalized as ligand binding sites

Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na+/H+ antiporter NHA2

Allosteric links between the hydrophilic N‐terminus and transmembrane core of human Na+/H+ antiporter NHA2

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Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na⁺/H⁺ antiporter NHA2

Allosteric links between the hydrophilic N‐terminus and transmembrane core of human Na⁺/H⁺ antiporter NHA2