Supplementary information

Transcription

1 Supplementary information The structural basis of modularity in ECF-type ABC transporters Guus B. Erkens 1,2, Ronnie P-A. Berntsson 1,2, Faizah Fulyani 1,2, Maria Majsnerowska 1,2, Andreja Vujičić-Žagar 1,2, Josy ter Beek 1,2, Bert Poolman 1,2 and Dirk Jan Slotboom 1,2 1 University of Groningen, Groningen Biomolecular Science and Biotechnology Institute, Nijenborgh 4, 9747 AG Groningen, the Netherlands. 2 University of Groningen, Zernike Institute for Advanced Materials, Nijenborgh 4, 9747 AG Groningen, the Netherlands. Supplementary information comprises: Supplementary Figures 1 8 Supplementary Table 1 Supplementary References 1

2 Supplementary Figure 1. The orientation of ThiT in the membrane. The orientation of ThiT in the membrane was deduced from the distribution of positive charges (positive inside rule 2 ). The positively charged amino acids (Lys, Arg) are colored blue; all other amino acids are depicted in gray. 2

3 Supplementary Figure 2. Sequence alignment of L. lactis ThiT with orthologs from various bacterial species. The position of the transmembrane helices in L. lactis ThiT is indicated above the sequences. Conserved residues not involved in ligand binding (e.g. structurally important residues) are colored green. The amino acids that interact directly with thiamin are highlighted in blue. Residues forming the network of hydrogen bonds and aromatic interactions around the substrate are colored yellow. The mechanistically important L1 loop is indicated by the purple bar. 3

4 Supplementary Figure 3: Schematic representation of the interactions in the high affinity thiamin binding site. Hydrogen bonds are depicted as red dashed lines and aromatic ring-stacking is indicated by blue dashed semi-circles. An ordered water molecule is represented by the black asterisk (*). 4

5 << helix 1 >> ThiT ---MSNSKFNVRLLTEIAFMAALAFIISLIPNTVYG-- 33 RibU MSKTRRMVLIAMLAALSTILLL-PILQFPL- 29 BioY ----MTNNQKVKTLTYSAFMTAFIIILGFLPGIPIGF- 33 PanT -----MKKSKASDVAILAIFIAIMVVVQLFTQFVINV- 32 HmpT --MKLMDNKNIKKLTLLAIWTALTFVLGRLFTFPI QeuT -----MKKSKTYDIVTIAIVAALYVILTMTPGLSAIS- 32 NiaX TQMTQTKKAKVRNLIIAAMLTALGILIPMMMPVKLIIG 38 BioY2 ----MQN-TKLYSLTLIALGAAIIAVLSPLA-IPIGI- 31 : : *: *: :: Supplementary Figure 4. Multiple-sequence alignment of the N-terminal residues containing the first transmembrane helix from all S-components that interact with the same energizing module in L. lactis. The amino acids in the first transmembrane helix of ThiT are shaded with a blue background. Highlighted in red is an alanine motif that is exposed to the predicted EcfT interface in ThiT. We found that the alanine motif is moderately conserved in sequence alignments of all S-components with their orthologs (not shown). 5

6 Supplementary Figure 5. Thiamin binding to WT ThiT and mutants. The assay was performed as described 3 using substrate free ThiT expressed in L. lactis. The dissociation constant values (inset) were determined by fitting the data to the binding equation as described 3. 6

7 Walker A EcfA MNKILEVENLVFKYEKES--DVNQLNGVSFSITKG-EWVSIIGQNGSGKSTTARLIDGLF 57 EcfA' ---MIKFEKVNYTYQPNSPFASRALFDIDLEVKKG-SYTALIGHTGSGKSTLLQHLNGLL 56 MetN ----MIKLSNITKVFHQGTRTIQALNNVSLHVPAG-QIYGVIGASGAGKSTLIRCVNLLE 55 ModC_AF MFLKVRAEKRLGNFRLN----VDFEMGRDYCVLLGPTGAGKSVFLELIAGIV 48 ModC_MA MIEIESLSRKWKNFSLDNLS-LKVESG-EYFVILGPTGAGKTLFLELIAGFH 50 MalK MASVQLQNVTKAWGEVVVSKDINLDIHEG-EFVVFVGPSGCGKSTLLRMIAGLE 53 BtuD MSIVMQLQDVAESTRLGPLSGEVRAG-EILHLVGPNGAGKSTLLARMAGMT 50.. *..:*.*.**: : : Q-loop EcfA EEFEGIVKIDGERLTAEN----VWNLRRKIGMVFQNPDNQFVGATVEDDVAFGMENQGIP 113 EcfA' QPTEGKVTVGDIVVSSTSKQKEIKPVRKKVGVVFQFPESQLFEETVLKDVAFGPQNFGIP 116 MetN RPTEGSVLVDGQELTTLS-ESELTKARRQIGMIFQHFNLL-SSRTVFGNVALPLELDNTP 113 ModC_AF KPDRGEVRLNGADITPLP------PERRGIGFVPQDYALF-PHLSVYRNIAYGLRNVERV 101 ModC_MA VPDSGRILLDGKDVTDLS------PEKHDIAFVYQNYSLF-PHMNVKKNLEFGMR-MKKI 102 MalK TITSGDLFIGEKRMNDTP------PAERGVGMVFQSYALY-PHLSVAENMSFGLKLAGAK 106 BtuD S-GKGSIQFAGQPLEAWS---ATKLALH-RAYLSQQQ------TPPFATPVWHYLTLHQH 99 * :. : : : * Signature motif Walker B EcfA REEMIKRVDEALLAVNMLD-FKTREPARLSGGQKQRVAVAGIIALRP EIIILD 165 EcfA' KEKAEKIAAEKLEMVGLADEFWEKSPFELSGGQMRRVAIAGILAMEP EVLVLD 169 MetN KDEVKRRVTELLSLVGLGD-KHDSYPSNLSGGQKQRVAIARALASNP KVLLCD 165 ModC_AF ERDR--RVREMAEKLGIAH-LLDRKPARLSGGERQRVALARALVIQP RLLLLD 151 ModC_MA KDPK--RVLDTARDLKIEH-LLDRNPLTLSGGEQQRVALARALVTNP KILLLD 152 MalK KEVINQRVNQVAEVLQLAH-LLDRKPKALSGGQRQRVAIGRTLVAEP SVFLLD 158 BtuD DKTRTELLNDVAGALALDD-KLGRSTNQLSGGEWQRVRLAAVVLQITPQANPAGQLLLLD 158 : : :.. ****: :** :. :. ::: * H-loop EcfA ESTSMLDPTGRSEIMRVIHEIKDKYHLTVLSITHDLDEAASSDRILVMRAGEIIKEAAP 224 EcfA' EPTAGLDPKARIEMMQLFESIHQS-GQTVVLVTHLMDDVADYADYVYLLEKGHIISCGTP 228 MetN EATSALDPATTRSILELLKDINRRLGLTILLITHEMDVVKRICDCVAVISNGELIEQDTV 225 ModC_AF EPLSAVDLKTKGVLMEELRFVQREFDVPILHVTHDLIEAAMLADEVAVMLNGRIVEKGKL 211 ModC_MA EPLSALDPRTQENAREMLSVLHKKNKLTVLHITHDQTEARIMADRIAVVMDGKLIQVGKP 212 MalK QPLSNLDAALRVQMRIEISRLHKRLGRTMIYVTHDQVEAMTLADKIVVLDAGRVAQVGKP 218 BtuD EPMNSLDVAQQSALDKILSALCQQ-GLAIVMSSHDLNHTLRHAHRAWLLKGGKMLASGRR 217 :. :* : : :: :*.. :: *.: EcfA SELFATSEDMVEIGLDVP FSSNLMKDLRTNG EcfA' SDVFQEVDFLKAHELGVP KATHFADQLQKTG MetN SEVFSHPKTPLAQKFIQS----TLHLDIPEDYQERLQAEP ModC_AF KELFS-AKNGEVAEFL SARNLLLKVSK---ILD ModC_MA EEIFEKPVEGRVASFVGFENVLKGRVISAEQGLLRIRVGEVVIDAAGDMEVG---DQVYA 269 MalK LELYHYPADRFVAGFIG SPKMNFLPVKVTATAIDQVQVELPMPNRQQVWL 268 BtuD EEVLTPPNLAQAYGMN FRRLDIEG :: : : Supplementary Figure 6. Sequence alignment of NBDs from ABC transporters with available structures and EcfA and A from L. lactis. The conserved ABC transporter motifs are: Walker A (P-loop) (orange), conserved glutamine of the Q-loop (blue), the ABC transporter signature motif (red), Walker B motif (yellow) and the H-loop (green). We have used two orthologs of ModC for this alignment: ModC from Archaeoglobus fulgidus (ModC_AF) and from Methanosarcina acetivorans (ModC_MA). The residues that line 7

8 the coupling helix binding groove are colored purple. These residues are remarkably conserved between EcfA and EcfA, and MetN, ModC, MalK and (to lesser extent) BtuD, from which structures were used to locate the groove, strongly suggesting that both EcfA and EcfA interact with a coupling helix, presumably present in the long cytoplasmic loop of EcfT as discussed in the main text. 8

9 Supplementary Figure 7. Predicted membrane topology of EcfT from L. lactis (gi: ). The cytoplasmic domain between helices 4 and 5 contains two moderately hydrophobic segments that could form coupling helices. The length of this domain could comfortably accommodate two coupling helices. The lipid membrane is indicated by the black dashed line. 9

10 a b Supplementary Figure 8. The ThiT molecules in the asymmetric unit. (a) Both chains of ThiT are shown in ribbon representation and colored in a rainbow fashion, from the N-terminus blue to the C-terminus in red. Peaks for the Se atoms in the anomalous difference Fourier map, calculated between 48.0 and 2.9 Å and contoured at 5σ, are shown in a grey mesh. A total of four SeMet were visible in the map. SeMet at position 1 and 9 in the protein construct were in a disordered region of the protein. Peak heights are as follows: Mse68: 34.9σ and 28.0σ (in chain A and B, respectively), Mse17: 26.7σ and 26.3σ (in chain A and B, respectively). No noise peaks were present at a 5σ cut-off. (b) Experimental electron density, the density is show as a gray mesh 10

11 (2F o - F c map contoured at 1.5σ). The ThiT main chain is depicted as a ribbon representation and colored as in Fig

12 Supplementary Table 1. The intricate network of hydrogen bonds and aromatic interactions between binding site residues. The following types of interactions were found: Hydrogen Bond (HB), Salt Bridge (SB), Aromatic L-shaped conformation 1 (A L ), Aromatic T-shaped conformation 1 (A T ). Residue Interactions with Trp-34 Tyr-74 (A L ) Glu-38 Tyr-122 (HB), Lys-121 (SB) Tyr-74 Trp-34 (A L ), Tyr-85 (HB, to OH-group C-α) Leu-76 Gln-80 (HB) Gln-80 Glu-84 (HB), Leu-76 (HB, to backbone N) Glu-84 Trp-133 (HB), His-125(HB), Gln-80(HB) Tyr-85 Tyr-74 (HB) Lys-121 His-125 (HB), Glu-38 (SB) Tyr-122 Glu-38 (HB) His-125 Lys-121 (HB), Glu-84 (HB) Trp-133 Glu-84 (HB) Trp-138 Tyr-146 (A T ) Trp-141 Tyr-146 (A T ) Tyr-146 Trp-138 (A T ), Trp-141 (A T ) Ser-147 Asn-151 (HB) Asn-151 Ser-147 (HB) 12

13 Supplementary References 1. Burley, S.K. & Petsko, G.A. Aromatic-aromatic interaction: a mechanism of protein structure stabilization. Science 229, (1985). 2. Von Heijne, G. Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues. Nature 341, (1989). 3. Erkens, G.B. & Slotboom, D.J. Biochemical characterization of ThiT from Lactococcus lactis: a thiamin transporter with picomolar substrate binding affinity. Biochemistry 49, (2010). 13