Mode of Interaction of the Signal-Transducing Protein EIIAGlc with the

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Mode of interaction of the signal-transducing protein EIIAGlc with the maltose ABC transporter in the process of inducer exclusion Steven Wuttge, Anke Licht, M. Hadi Timachi, Enrica Bordignon, and Erwin Schneider Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00721 • Publication Date (Web): 29 Aug 2016 Downloaded from http://pubs.acs.org on August 30, 2016

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Biochemistry

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Mode of interaction of the signal-transducing protein EIIAGlc with the

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maltose ABC transporter in the process of inducer exclusion

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Steven Wuttge1§$, Anke Licht1§, M. Hadi Timachi2&, Enrica Bordignon2&*, and

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Erwin Schneider1*

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1

8

Linden 6, 10099 Berlin, Germany

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2

Fachbereich Physik, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany

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$

Present address: Beyontics, Altonaer Str. 79-81, 13581 Berlin, Germany

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&

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Universitätsstraße 150, 44801 Bochum, Germany

Institut für Biologie, AG Bakterienphysiologie, Humboldt-Universität zu Berlin, Unter den

Present address: Faculty of Chemistry and Biochemistry, Ruhr-Universität Bochum,

13 14 15

§

These authors contributed equally to this work

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*To whom correspondence should be addressed:

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Erwin

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Schneider:

Phone:

+49(30)209349780.

Fax:

+49(30)209349781.

E-Mail:

[email protected] Enrica Bordignon: Phone: +49(234)3226239. E-mail: [email protected]

21 22

1

ACS Paragon Plus Environment

Biochemistry

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Funding information: This work was supported by the Deutsche Forschungsgemeinschaft

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(SCHN 274/15-1, 2 to E.S.), (BO 3000/1-2 to E.B.) and by the Cluster of Excellence

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RESOLV (EXC 1069).

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Abbreviations: ABC, ATP-binding cassette; CuPhe, Cu(1,10-phenanthroline)2SO4; DEER,

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double electron electron resonance; DDM, n-dodecyl-β-D-maltopyranoside; EBS, 1,2

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ethanediyl-bismethanethiosulfonate; EPR, electron paramagnetic resonance spectroscopy;

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HBS, 1,6 hexandiyl-bismethanethiosulfonate; His-tag, hexahistidine tag; IPTG, isopropyl-β-

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D-thio-galactopyranoside;

MTSL,

(1-Oxyl-2,2,5,5-tetramethylpyrrolidin-3-yl)

methyl

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methanethiosulfonate label; NBD, nucleotide binding domain; NEM, N-ethylmaleimide; Ni-

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NTA, Nickel-nitrilotriacetic acid; OG, octyl-ß-D-glucopyranoside; PBS, 3,6,9,12,15-

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pentaoxaheptadecan-1,17-diyl-bis-methanethiosulfonate;

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phenylmethylsulfonylfluoride; PTS, phosphotransferase system; SBP, solute binding protein;

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TMD, transmembrane domain;

PMSF,

15

2


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Abstract

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Enzyme IIAGlc (EIIAGlc) of the phosphoenolpyruvate phosphotransferase system for the

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uptake of glucose in Escherichia coli/Salmonella inhibits the maltose ATP-binding cassette

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transporter (MalE-FGK2) by interaction with the nucleotide-binding and -hydrolyzing subunit

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MalK, a process termed inducer exclusion. We have investigated binding of EIIAGlc to the

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MalK dimer by cysteine cross-linking in proteoliposomes. The results prove that the binding

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site I of EIIAGlc is contacting the N-terminal subdomain of MalK while the binding site II is

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relatively close to the C-terminal (regulatory) subdomain, in agreement with a crystal

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structure [Chen, S., Oldham, M.L., Davidson, A.L., and Chen, J. (2013) Nature 499, 364-

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368]. Moreover, EIIAGlc was found to bind to the MalK dimer regardless of its conformational

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state. Deletion of the amphipathic N-terminal peptide of EIIAGlc, which is required for

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inhibition, reduced formation of cross-linked products. Using a spin-labeled transporter

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variant and EPR spectroscopy we demonstrate that EIIAGlc arrests the transport cycle by

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inhibiting the ATP-dependent closure of the MalK dimer.

16 17

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Biochemistry

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The transporter mediating the uptake of maltose and maltodextrins in Escherichia

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coli/Salmonella is one of the best characterized members of the superfamily of ATP-binding

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cassette (ABC)-transport systems. ABC transporters comprise two transmembrane domains

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(TMDs) that form the translocation pathway and two nucleotide-binding domains (NBDs)

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that bind and hydrolyze ATP1, 2. ABC transporters are thought to operate by an ‘alternate

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access’ mode with the translocation path shuttling between an inward-facing and outward-

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facing conformation in response to substrate and ATP binding and hydrolysis3. Canonical

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ABC importers confined to prokaryotes also depend on extracellular (or periplasmic) solute

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binding proteins (SBP), playing a crucial role in initial steps of the transport cycle4, 5.

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The maltose transporter consists of the periplasmic maltose binding protein, MalE, one

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copy each of the membrane-integral subunits, MalF and MalG, and two copies of the ATPase

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subunit, MalK6, 7. In enteric bacteria, such as Escherichia coli and Salmonella enterica

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serovar Typhimurium (S. Typhimurium), the activity of the maltose transporter and other

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sugar permeases is controlled by the phosphoenolpyruvate carbohydrate phosphotransferase

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system (PTS), a key player in global carbon regulation8. The PTS comprises a cascade of

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protein kinases and phosphocarriers that constitute a series of transport systems which couple

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transport and phosphorylation of numerous sugars. The pathway requires the sequential

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transfer of a phosphoryl group from phosphoenolpyruvate via enzyme I (EI) to the histidine

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protein (HPr) and finally to the sugar-specific enzyme II (EII) components. 'Catabolite

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repression' allows for the preferential utilization of so-called Class A-sugars (e.g. glucose),

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when cells are cultured in the presence of other -Class B- sugars. The PTS regulates the

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uptake of Class B-sugars by the phosphorylation state of the glucose-specific EIIAGlc

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component playing a key role in this process. Phosphorylated EIIAGlc is involved in the 4


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activation of adenylate cyclase which produces cAMP, a crucial regulatory molecule for

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controlling the expression of numerous genes the products of which are involved in the

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metabolism of Class B-sugars9. In contrast, when glucose is the preferred carbon source,

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EIIAGlc becomes predominantly unphosphorylated and inhibits uptake of maltose, melibiose,

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lactose, glycerol and other carbohydrates by direct binding to the permeases10-12 or glycerol

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kinase13. EIIAGlc is phosphorylated by HPr-P at His-9014 which is largely buried within a ring

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of solvent exposed hydrophobic residues that have been suggested to provide a binding site

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for (regulatory) targets10, 13, 15-18. Inhibition of maltose transport by EIIAGlc in vivo8 is caused

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by eliminating substrate-stimulated ATPase activity of the transporter through direct

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interaction with the MalK subunits as demonstrated in vitro10, 19-21. Unlike nucleotide-binding

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domains of most other ABC transporters, MalK contains a C-terminally located ‘regulatory’

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subdomain that forms a large part of the dimer contact surface22,

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structure of the full transporter in complex with two molecules of EIIAGlc revealed that this

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subdomain preferentially interacts with EIIAGlc

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previous findings that (i) most mutations rendering the transporter insensitive to inducer

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exclusion localize to the C-terminal subdomain of MalK19,

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monoclonal antibody recognizing a C-terminal peptide of MalK was reduced by EIIAGlc

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Moreover, the structural data revealed, as proposed previously27, that one molecule of EIIAGlc

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interacts with amino-terminal residues of one MalK monomer and the C-terminal subdomain

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of the opposing monomer.

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(Fig. 1A). The crystal

10

. This observation is consistent with

24, 25

, and (ii) binding of a 26

.

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The crystal structure also corroborated our previous proposal based on a study employing

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synthetic peptide arrays combined with mutational analysis28 that two sites on EIIAGlc are

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involved in contacting MalK. Site I (including residues 69-79 and 87-91) partly overlaps but 5


Biochemistry

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is not identical to that for interaction with glycerol kinase13 and lactose permease29, while site

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II (residues 118-127) is MalK-specific (Fig. 1B).

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Furthermore, the N-terminal peptide of EIIAGlc was demonstrated to be crucial for

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inhibition of transporter activity10, 21, 28. The N-terminal tail which is not resolved in any of the

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known crystal structures, was suggested to confer amphitropism to EIIAGlc, allowing the

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protein to shuttle between the cytoplasm and the membrane30, 31.

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During the translocation cycle, the MalK dimer switches from an open (cofactor-less) apo-

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state to a closed state upon binding of ATP and liganded MalE and returns to the apo-state

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following ATP hydrolysis and dissociation of phosphate and ADP6, 7. Based on a crystal

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structure of the transporter residing in the apo-state in the absence of MalE and complexed to

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EIIAGlc, Chen et al.10 proposed a model according to which EIIAGlc prevents the MalK dimer

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from closing. However, whether EIIAGlc binds exclusively to the apo-state of the transporter

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cannot be deduced from the frozen snapshot of the crystal structure. Biochemical evidence

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provided by Bao and Duong21 demonstrating formation of less ADP in the presence of EIIAGlc

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compared to the control also led to the suggestion that inhibition of ATPase activity by

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EIIAGlc is due to blocked MalK dimer closure. However, no direct evidence for this

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hypothesis was thus far provided.

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To address these questions, we performed site-specific cross-linking of single cysteine

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variants combined with pulsed EPR techniques on spin-labeled variants (Double Electron

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Electron Resonance, DEER) to investigate the interaction of EIIAGlc with the MalK dimer in

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the context of the reconstituted transporter in the presence of MalE/maltose. We show that

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EIIAGlc is contacting the MalK subunits of the transporter in all steps of the nucleotide cycle.

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Furthermore, distance distributions of spin-labeled residues V17C and E128C in MalK 6


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determined by DEER provided first direct evidence that EIIAGlc inhibits the transporter by

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preventing the ATP-induced closure of the NBDs32, 33.

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Experimental procedures

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Bacterial strains, plasmids, and media

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All strains and plasmids used in this study are listed in Table S1. The plasmid-borne malK

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alleles originate from S. Typhimurium, whereas the malFG alleles on pTZ18R encoding cys-

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less variants are from E. coli. The mal genes from both organisms are functionally fully

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exchangeable34. E. coli strain JM109 served as host for general cloning purposes and for the

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expression of maltose transporter variants. EIIAGlc variants were overproduced in E. coli

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strain BL21(DE3)∆(pts43crr::kanR)26 lacking HPr and therefore preventing phosphorylation

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of the proteins. Bacteria were usually grown in LB35 or TPi36 medium, supplemented with

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ampicillin (100 µgml-1) and/or chloramphenicol (20 µg ml-1) if required.

13

Site-directed mutagenesis

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Derivatives of plasmid pBB04 (crr+)28 and pMM37 [malK796 (C40S)]37 carrying single

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point mutations were obtained by site-directed mutagenesis using Stratagene’s QuikChange

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kit.

17 18

Protein preparations

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Polyhistidine-tagged maltose transporter variants for cross-linking experiments were

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purified from cells of strain JM109 harboring plasmid pMM34 (MalF*G*, * denotes cysless

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subunits) in combination with pMM37 [MalK(C40S)2], pBK02 [MalK(C40S, Q122C)2],

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pBK04 [MalK(C40S, R322C)2], pBK05 [MalK(C40S, A320C)2], and pHL09 [MalK(C40S,

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E119C)2], respectively, as described in Ref. [37]. Briefly, proteins solubilized from membrane 7


Biochemistry

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vesicles by addition of 1.1 % DDM were bound to Ni-NTA resin equilibrated in buffer A (50

2

mM Tris-HCl, pH 7.5, 20 % glycerol, 0.1 mM PMSF, 0.01 % DDM). The resin was washed

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with buffer A, supplemented with 20 mM imidazole, and protein was eluted with buffer A

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supplemented with 200 mM imidazole. Peak fractions were pooled, passed through a PD10

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column (GE Healthcare) equilibrated with buffer B (50 mM Tris-HCl, pH 7.5, 10 % glycerol,

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0,01 % DDM), concentrated by ultrafiltration, shock-frozen in liquid nitrogen, and stored at -

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80 °C. The ATPase activities of all transport complexes reconstituted in proteoliposomes are

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listed in Table S3

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The transporter variant used for DEER measurements was prepared likewise from cells

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harboring plasmids pMM34 (MalF*G*, cysless) and pAL66 [MalK(C40S, C350M, C360M,

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V17C, E128C]. Spin labeling of the transporter mutant with MTSL was performed as

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described elsewhere38. ATPase activities of the transporter embedded in nanodiscs are listed

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in Table S5.

14 15

Polyhistidine-tagged MalE was purified from cells of strain JM109(pCB6) as described in Ref. [39].

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EIIAGlc variants were purified from strain BL21(DE3)∆pts harboring plasmids

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pBB04(wild type), pHL04(P125C), pWS02(K69C), pWS08(∆1-16, P125C), pWS09(E97C),

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pWS19(∆1-16, K69C), pWS29(E160C), and pBB04(F88Q), respectively, as in Ref. [28]. The

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inhibitory potential of all EIIAGlc variants on the ATPase activity of maltose transport

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complexes considered in this study are listed in Tables S4 and S5 .

21

Preparation of proteoliposomes

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For cross-linking experiments, maltose transporter variants were incorporated into

23

liposomes at a 10:1 lipid to protein ratio (E. coli total lipid extract, Avanti Polar Lipids, 8


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Alabaster, USA). Typically, 250 µg of complex protein were mixed with 2.5 mg

2

phospholipids dissolved in 50 mM Tris-HCl, pH 7.5, 1 % OG. Reconstitution using Biobeads

3

(BioRad, Munich, Germany) was performed overnight at 4°C in the presence and absence of

4

MalE/maltose (10 µM/60 mM) in a total volume of 300 µl. After replacing the beads with a

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fresh batch incubation continued for 1 h. The mixture was subsequently centrifuged at low

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speed to remove the beads, followed by a spin at 200.000 x g for 30 min at 4°C to collect the

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proteoliposomes which were then resuspended in 200 µl of 50 mM Tris-HCl, pH 7.5, 10 µM

8

maltose. For ATPase activity measurements, proteoliposomes were prepared at a lipid to

9

protein ratio of 50:1 essentially as described elsewhere32 .

10

Preparation of nanodiscs

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Nanodiscs containing MalF*G*K*2 or its variants were prepared as described in Ritchie et

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al.40 using the membrane scaffold protein MSP1E3D1 and the solubilized E. coli total lipid

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extract (Avanti Polar Lipids). Lipid was dried in a rotating vacuum evaporator, resuspended

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in 20 mM Tris/HCl (pH 7.5), 0.1 M NaCl, 1% DDM and the mixture was sonicated for 2 min

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in a bathtype sonicator. Complex protein, MSP and the lipid preparation were mixed at a

16

molar ratio of 1:4:480. Buffer was added to reduce the glycerol concentration to below 3%,

17

and the mixture was incubated at 4 °C for 1 h. Reconstitution was performed by adding Bio-

18

Beads (200 mg/1 mg complex, Bio-Rad) for 3 h at 4 °C under gentle shaking. Bio-Bead

19

removal was followed by the isolation of the assembled complex containing nanodiscs by

20

metal-affinity chromatography using a HisPur™ Ni-NTA resin (Thermo Scientific) in the

21

presence of 50 mM Tris/HCl (pH 7.5), 50 mM NaCl. After washing the resin with buffer,

22

protein was eluted with 250 mM imidazole. Protein-containing fractions were pooled,

23

concentrated, buffered in 50 mM Tris/HCl (pH 7.5) by passage through a PD10 desalting 9


Biochemistry

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column (GE Healthcare) and stored at 4°C. The concentration was determined by pixel

2

analysis following SDS PAGE. Since two MSP molecules form a single nanodisc, an MSP to

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MalK ratio of 1:1, as inferred by the pixel analysis, indicates that one MalFGK2 complex was

4

present per nanodisc.

5

Cross-linking

6

Cross-linking experiments using CuPhe or homobifunctional thiosulfonate linkers were

7

performed as described in Ref. [41]. Thiosulfonate cross-linkers were purchased from

8

Toronto chemicals (Toronto, Canada). The following cross-linkers (approximate spacer

9

lengths in parenthesis) were used: EBS (5.2 Å), HBS (10.4 Å), and PBS (24.7 Å)42.

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Proteoliposomes and EIIAGlc variants were incubated in 50 mM Tris-HCl, pH 7.5, 10 µM

11

maltose for 5 min at room temperature in the absence of cofactors and in the presence of

12

ATP/EDTA (4 mM/0.1 mM) or ADP (4 mM). Samples containing ATP/Mg2+ (4 mM/4 mM)

13

were incubated for 10 min at 37 °C. Cross-linking reactions were started by adding CuPhe (3

14

mM CuSO4 and 9 mM 1,10-phenanthroline) or thiosulfonate linkers (final concentration 1

15

mM) from freshly prepared stock solutions (100 mM in dimethylsulfoxide). If not stated

16

otherwise, reactions were terminated after 20 min at room temperature by adding 5 mM

17

NEM.

18

DEER experiments

19

DEER experiments were performed on the transporter variant MalF*G*K(V17R1,

20

E128R1)2 (~ 37 µM final concentration, labeling efficiency calculated to be 120%) embedded

21

in nanodiscs in the absence or presence of 12-fold molar excess of EIIAGlc under three

22

different experimental conditions: absence of nucleotides (apo-state), incubation with 3 mM

23

ATP and 0.075 mM EDTA to prevent hydrolysis (ATP-state), and incubation for 5 minutes at 10


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37°C with 3 mM ATP and 5 mM MgCl2 to reach the post-hydrolysis state. All samples

2

contained maltose-loaded MalE at a 3-fold stoichiometric ratio with respect to the transporter

3

and 10% (vol/vol) d8-glycerol. Two additional control experiments were performed; i) an

4

EIIAGlc mutant (F88Q) which lacks inhibitory function was used in the presence of ATP-

5

EDTA to monitor if the structural response was different, and ii) EIIAGlc was added after

6

incubation of the sample with ATP-EDTA to investigate the reversibility of the effects

7

observed.

8

Double electron-electron resonance (DEER) measurements were performed at 50 K on a

9

Bruker ELEXSYS E580Q-AWG (arbitrary wave generator) dedicated pulse Q-band

10

spectrometer equipped with a 150 W TWT amplifier and a home-made resonator for tubes

11

with 3 mm outer diameter. A 4-pulse DEER sequence with rectangular, non-selective pulses

12

of 16 ns length (for both π/2 and π pulses in the observer and pump frequencies) with 100

13

MHz frequency separation was used43. Due to coherent nature of the AWG generated pulses,

14

a four-step phase cycling of the pump π pulse was performed to completely remove the

15

effects of running echoes from the DEER trace. The background of the DEER primary data

16

[V(t)] was fitted and the resulting secondary data [F(t)] were converted by a model-free

17

Tikhonov regularization to distance distributions with the software DeerAnalysis201544.

18

Validation of the reliability of distance distributions was performed with the validation tool in

19

DeerAnalysis2015. The distance distribution in the 1.5-4 nm range, which allows monitoring

20

the vicinity of the NBDs, was found to be highly reproducible. MalK2 is a symmetric

21

homodimer; thus, spin-labeling of the double cysteine mutants (V17C–E128C) in the

22

nucleotide-binding domains (NBDs) leads to a four-spin system with the 17–128′ (the prime

11


Biochemistry

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denotes the second MalK monomer) and the 128–17′ pairs characterized by short distances,

2

which clearly report the closure and reopening of the NBDs32.

3 4

Analytical Procedures

5

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), protein

6

determination and ATPase assay were performed as in37. Inhibition of ATPase activity by

7

EIIAGlc was assayed by incubation of MalE/maltose-loaded proteoliposomes with EIIAGlc

8

variants for 5 min at 37 °C prior to the addition of Mg2+/ATP (4 and 2 mM, respectively).

9 10

Results

11

Rationale and experimental set-up

12

The crystal structure of the maltose transporter residing in the (cofactor-less) apo-state in

13

the absence of MalE and in complex with two copies of EIIAGlc 10 largely confirmed previous

14

proposals6, 27 that the N-terminal subdomain of MalK-A is in close contact to binding site I of

15

EIIAGlc while binding site II interacts with the C-terminal subdomain of MalK-B (Fig. 1C).

16

Based on this structural information, we designed experiments aimed to elucidate whether the

17

presence of nucleotides affects the binding of EIIAGlc to MalK2 and how the contact sites

18

found in the ‘frozen’ crystal snapshot reflect EIIAGlc - transporter interactions in the different

19

nucleotide states in a lipid environment.

20

To this end, we performed site-specific cross-linking experiments with the transporter

21

incorporated in proteoliposomes using mono-cysteine variants of MalK and EIIAGlc. We

22

chose EIIAGlc variants K69C (site I), E97, and P125C (site II) (Fig. 1B, C). Residues in MalK

23

to be replaced by cysteines - E119 and A320 - were selected from the N- and C- terminal 12


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subdomains, respectively, previously shown to be crucial for sensitivity to EIIAGlc 19, 24. In

2

addition, we included Q122 and R322 not previously recognized to be critical for interaction

3

with EIIAGlc. While MalK-A(Q122) forms a hydrogen bond with His9010, the

4

phosphorylation site of EIIAGlc 14, R322 is in close contact to binding site II of EIIAGlc (Fig.

5

1A, C). The Cβ- Cβ distances determined from the X-ray structure predict the indicated

6

residues to be positioned within cross-linking distance (Table S3).

7

The transporter variants were purified and incorporated into proteoliposomes loaded with

8

MalE/maltose. Since the complex molecules are embedded in the lipid bilayer in two opposite

9

orientations20, these conditions assured that only those which expose the MalK subunits to the

10

medium side contributed to ATPase activity. As summarized in Table S2, all variants

11

exhibited MalE/maltose-dependent ATPase activity similar to that of the control complex

12

MalF*G*K(C40S)2. In the presence of wild type EIIAGlc (5-fold molar excess), ATPase

13

activity of the control complex was inhibited by ~ 73 %, in agreement with previous reports20,

14

28

15

and mutants), albeit somewhat less than the control (Table S4). In contrast, the mutation

16

MalK(R322C) caused complete resistance to EIIAGlc, thereby underscoring the above notion

17

(Table S4). The mutation probably disrupts a hydrogen bond with EIIAGlc(E128) that might

18

be crucial for function10. Some inhibition of ATPase activity (between 11-14 %) was still

19

observed in case of the complex containing MalK(A320C). Together, these results are

20

consistent with previous findings that most mutations causing resistance to EIIAGlc map to the

21

C-terminal subdomain of MalK19, 24, 25.

. Most other transporter variants also displayed substantial sensitivity to EIIAGlc (wild type

22 23

Interactions between the N-terminal subdomain of MalK and binding site I of EIIAGlc 13


Biochemistry

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1

In a first set of experiments meant to establish the experimental system and illustrated

2

schematically in Fig. 1D, proteoliposomes containing MalF*G*K(C40S, E119C)2 and loaded

3

with MalE/maltose were incubated with EIIAGlc(K69C) in the presence of the oxidant

4

Cu(1,10-phenanthroline)2SO4 (CuPhe). CuPhe catalyzes the formation of disulfide bonds in

5

the presence of oxygen45. Although CuPhe is designated a ‘zero-length’ linker, it is well

6

established that conformational flexibility can influence the apparent distance between

7

residues46, 47. Consequently, significant flexibility of a polypeptide chain can result in cross-

8

linking of residues that are further apart in the native structure, e.g. 10-30 Å as demonstrated

9

in Ref. [47].

10

Disulfide bond formation between MalK(E119C) and EIIAGlc(K69C) was subsequently

11

analyzed by SDS-PAGE under non-reducing conditions (Fig. 2A). A cross-linked product

12

was found migrating in between molecular mass standards of 66 and 97 kDa and

13

demonstrated by immunoblotting to react with antisera raised against MalK and EIIAGlc,

14

respectively (Fig. S1). In addition, protein bands migrating at an apparent molecular mass of

15

110 kDa and around 50 kDa were identified as dimers of MalK and EIIAGlc, respectively.

16

(Please note that the apparent molecular mass of EIIAGlc depends on the position of the

17

introduced cysteine residue as can be seen in Figs. 2B-D).

18

The experiment was repeated in the presence of ATP and ADP, thereby mimicking the pre-

19

and the post-hydrolysis states of the transporter, respectively. The (cofactor less) apo-state is

20

characterized by an open conformation of the MalK dimer, while the ATP- and ADP- states

21

by closed and semi-open conformations, respectively10,

22

subjected to ATP hydrolysis prior to the addition of CuPhe, thus it also represents the post-

48-51

. An additional sample was

14


Page 15 of 45

Biochemistry

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1

hydrolytic state. As shown in Fig. 2A (left panel), the intensity of the cross-linked product

2

remained largely unchanged under all conditions tested.

3

Shortening the incubation time with CuPhe from 20 min to 10 s did not change the

4

intensity of the cross-linked band, thus limiting the possibility that cross-linked products

5

accumulated over time (Fig. 2B). Additionally, to exclude that cross-links were formed by

6

simple collision between mono-cysteine containing partner proteins, we repeated the

7

experiment with only 1/5 of the concentration of EIIAGlc(K69C). As expected for a

8

concentration-dependent event, EIIAGlc(K69C) dimer formation was greatly reduced but not

9

the EIIAGlc(K69C) - MalK(E119C) cross-links indicating specificity of the reaction (Fig. 2B,

10

right panel). Furthermore, no cross-linked product was obtained with EIIAGlc(E160C) (Fig.

11

2C), a residue located opposite to the contact sites and 30 Å (Cβ- Cβ distance) apart from

12

MalK-A(E119) in the crystal structure (Figs. 1B, C; Table S2).

13

As shown in Fig. 2C, EIIAGlc (E160C) forms strong dimers in the presence of CuPhe

14

which might prevent cross-linking to MalK. However, when cross-linking was performed

15

with flexible homobifunctional thiosulfonate linkers with defined spacer lengths in the

16

extended conformation (EBS, 5.2 Å; HBS, 10.4 Å; PBS, 24.7 Å) (see ‘Experimental

17

procedures’ for details) (Fig. 2C, right panel), EIIAGlc dimers hardly occurred (as can be seen

18

by comparing the amount of monomeric EIIAGlc with that in the presence of CuPhe).

19

Nonetheless, a cross-linked product between MalK(E119C) and EIIAGlc(E160C) was not even

20

obtained with PBS, thus supporting the notion that the residue is not taking part in interaction

21

with MalK.

22

Together, we conclude that the relative distance between MalK(E119) and EIIAGlc(K69) is

23

not dependent on the conformational state of the transporter. The same holds for the cross15


Biochemistry

Page 16 of 45

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1

linked products formed between MalK(Q122C) and EIIAGlc residues K69C and E97V,

2

respectively (Fig. 2D).

3

Interactions between the C-terminal subdomain of MalK and binding site II of EIIAGlc

4

As shown in Table S4, A320 located in the C-terminal subdomain of MalK (Fig. 1A and

5

C) when replaced by cysteine rendered the transporter largely resistant to inhibition by

6

EIIAGlc. Nonetheless, we performed cross-linking experiments with this variant since the

7

observed phenotype does not necessarily exclude binding to EIIAGlc. In fact, as shown in Fig.

8

3A, a cross-linked product with EIIAGlc(P125C) was formed under all conditions applied,

9

again in agreement with the relative distance between both residues as determined from the

10

crystal structure (Table S3).

11

Experiments with the transporter variant containing MalK(R322C) although fully resistant

12

to inhibition by EIIAGlc confirmed these results (Fig. 3B), thereby adding to the notion that

13

the mutation does not eliminate binding to MalK as such.

14

Cross-linking EIIAGlc to MalK is reduced in the absence of the N-terminal amphipathic

15

peptide

16

As shown previously, inhibition of ATPase activity of the maltose transporter by EIIAGlc

17

requires the N-terminal peptide10,

21, 28

18

phospholipid bilayer30,

19

binding of EIIAGlc to MalK, we performed cross-linking with EIIAGlc variants lacking

20

residues 1-16. As shown in Fig. 2A, cross-linked products were formed with

21

MalF*G*K(E119C)2 and EIIAGlc∆1-16(K69C), but to a minor extent with respect to the full-

22

length protein. The same holds for cross-linking MalF*G*K(A320C)2 with EIIAGlc∆1-

23

16(P125C) (Fig. 3A). We conclude that the N-terminal peptide of EIIAGlc, known to be

which is thought to attach the protein to the

31

. To address the question whether this peptide is also crucial for

16


Page 17 of 45

Biochemistry

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1

crucial for inhibition, seems to be strongly required for correct positioning of both binding

2

sites at the complex.

3

EIIAGlc inhibits ATP-dependent MalK dimer closure

4

The cross-linking data suggest that EIIAGlc binds to the MalK dimer regardless of its

5

conformational state. If so, how does EIIAGlc interfere with the ATPase cycle? To address this

6

question we analyzed whether EIIAGlc when bound to the apo-state of the transporter might

7

prevent the ATP-induced closure of the MalK dimer which triggers the transition of the

8

transmembrane domains from an inward-facing to an outward-facing conformation together

9

with MalE/maltose. To this end, we performed pulsed EPR (DEER) measurements with a

10

spin-labeled transporter variant (MalKV17R1, E128R1) already used in a previous study32

11

and embedded in nanodiscs (see Experimental procedures for details). Nanodiscs are

12

composed of a phospholipid bilayer domain with two copies of an amphipathic membrane

13

scaffold protein (MSP) that wrap around the periphery of the bilayer. They are formed by

14

removal of detergent from initial detergent-phospholipid micelles. When a membrane protein

15

is present, it is trapped within the forming nanodisc structure40. The advantage over

16

proteoliposomes lies in the accessibility of the membrane protein from both sides of the

17

bilayer, which allows that 100% of the frozen transporters are in contact with the nucleotides,

18

facilitating data analysis. The 17–128′ (the prime denotes the second MalK monomer) and the

19

128–17′ distances are distinctly shorter than all other intra- and inter-MalK distances and

20

report closure and reopening of the NBDs, according to the crystal structures and previous

21

DEER data32. The spin-labeled variant displayed MalE/maltose-stimulated and EIIAGlc-

22

sensitive ATPase activity comparable to that of MalF*G*K*2 (Table S5).

17


Biochemistry

Page 18 of 45

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1

As shown in Fig. 4A, the apo-state of MalFGK2 (in the presence of MalE/maltose which

2

was added to all samples) showed a distance peak centered at 3.5 nm (Fig. 4A, cyan trace),

3

which was associated with the 17–128′ and 17′–128 distances. Addition of ATP/EDTA

4

shifted the distance peak to 2.2 nm (Fig. 4A, dark green) indicating closure of the MalK

5

dimer. Upon hydrolysis of ATP in the presence of Mg2+-ions, a semi-open conformation of

6

the NBDs with a mean interspin distance of about 3 nm was found (Fig. 4A, red). These data

7

are perfectly in line with previous findings that were obtained with the transporter in detergent

8

solution32. Repeating these measurements in the presence of EIIAGlc clearly demonstrated that

9

the open apo- and semi-open post-hydrolytic conformations of the NBDs are mostly

10

unaffected by the presence of EIIAGlc (Fig. 4B, cyan and red). However, the ATP-

11

MalE/maltose-dependent closure of the MalK dimer was inhibited. In fact, the samples

12

preincubated with EIIAGlc showed in the presence of ATP-EDTA a distance distribution

13

largely dominated by an apo-like distance of 3.5 nm (Fig. 4B, dark green). Only a small

14

fraction of transporters in the frozen molecular ensemble are trapped with closed NBDs (2 nm

15

distance). Strikingly, when the transporter was incubated with ATP/EDTA prior to addition of

16

EIIAGlc, thus allowing the NBDs to close before binding to EIIAGlc, we detected a distance

17

distribution centered at 2.2 nm, typical for closed NBDs (compare Fig. 4A dark green and 4C

18

light green). This proves that EIIAGlc inhibits the closure of the NBDs but it is unable to re-

19

open a closed MalK dimer.

20

As control, we monitored the effects of the EIIAGlc(F88Q) variant which has lost the

21

inhibitory function28. When this dysfunctional variant was present, adding ATP-EDTA to the

22

sample induced a complete closure of the MalK dimer (compare Fig. 4A, dark green and Fig.

23

4C, brown). This finding proves that the effects observed with the native EIIAGlc in the ATP18


Page 19 of 45

Biochemistry

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1

EDTA sample are relevant for its mechanism of action, namely preventing the substrate- and

2

nucleotide-dependent closure of the MalK dimer.

3 4

DISCUSSION

5

We have addressed the question of how the unphosphorylated form of the PTS component

6

EIIAGlc interacts with the MalK subunits of the maltose ABC transporter in the process of

7

inducer exclusion via cross-linking methods and DEER spectroscopy.

8

Our results from cross-linking analysis performed with proteoliposomes, which provide a

9

lipid environment crucial for monitoring functional interaction of both partners, revealed no

10

preference of EIIAGlc for a distinct conformational state of the transporter in terms of binding.

11

This indicates that the contact sites on the MalK dimer remain largely unchanged during its

12

transition from the apo-(inward-facing) state through the ATP-bound (outward-facing) and to

13

the ADP-bound (semi-open, post-hydrolysis) state. This notion is in line with structural

14

evidence demonstrating no significant conformational alterations of the C-terminal

15

subdomains of the MalK dimer between the apo- and the ATP-bound state of the transporter48,

16

50, 52

17

their relative positions to the MalK dimer depending on the nucleotide status within distances

18

that cannot be distinguished by cross-linking.

. Nonetheless, our data do not exclude that binding sites I and II of EIIAGlc might change

19

Is this lack of sensing a particular conformation of MalK2 physiologically meaningful? In

20

case of MalT, the positive transcriptional regulator of the mal regulon, which also interacts

21

with the C-terminal extension of MalK, it was previously shown that binding occurs to the

22

resting state only53. This makes sense as binding of MalT to the transporter in the absence of

23

substrate prevents its activation by oligomerization and thus transcription of the mal genes. 19


Biochemistry

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1

In contrast, in the presence of a preferred energy source such as glucose, EIIAGlc must

2

prohibit uptake of maltose and maltodextrins by the maltose transport complexes to be

3

utilized as alternative substrates and/or inducers for mal gene expression. Thus, it appears

4

reasonable to assume that this goal is best achieved by binding to all possible conformational

5

states of the transporter molecules.

6

Based on the crystal structure that was obtained in the absence of cofactors and MalE,

7

Chen et al.10 proposed a model according to which EIIAGlc arrests the transporter in its apo-

8

state. However, the (cofactor-less) apo-state is unlikely to stably exist in the cell since the

9

cytoplasmic concentration of ATP is in the millimolar range54-56 and thus well above the Km

10

for ATP determined with the purified complex (74 µM)57. Thus, a model by which EIIAGlc

11

arrests the transporter in the apo-state to explain inducer exclusion under physiological

12

conditions was at least questionable. Subsequently, Bao and Duong21 by investigating the

13

maltose transporter embedded in nanodiscs, demonstrated that neither binding of the

14

fluorescent analog TNP-ATP nor release of ADP upon ATP hydrolysis was affected by

15

EIIAGlc. Furthermore, they provided evidence that in the presence of EIIAGlc less ADP is

16

produced, proof that EIIAGlc inhibits ATP hydrolysis. The authors speculated that this might

17

be achieved by holding the MalK dimer in its open conformation even when ATP is bound.

18

The data presented in this communication provide direct evidence that indeed EIIAGlc

19

prevents the MalK dimer from closing, but cannot reopen a sandwiched NBD dimer.

20

Moreover, binding of EIIAGlc to the transporter regardless of its conformational state as

21

demonstrated by cross-linking assures that it can readily interfere after dissociation of the

22

hydrolysis products and before re-binding of ATP. Putting together all available evidence led

23

us to propose the following model (Fig. 5): In the absence of EIIAGlc, the transporter switches 20


Page 21 of 45

Biochemistry

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

from an inward facing (open MalK dimer) (I) to an outward facing conformation (closed

2

MalK dimer) upon binding of ATP and maltose-loaded MalE (II)32, 33. Maltose is released into

3

the translocation path48 and, following ATP hydrolysis, delivered to the cytoplasm (III). After

4

dissociation of ADP which keeps the MalK dimer in a semi-open state49 (III) the transporter

5

returns to the inward-facing conformation. As shown in this study, EIIAGlc can interact with

6

the transporter residing in any of these conformations. Binding to the inward-facing

7

conformation locks the MalK dimer in the open state10 (A), unable to close upon addition of

8

ATP and MalE/maltose (B). When EIIAGlc binds to the outward-facing conformation (C), it

9

does not re-open the MalK dimer (Fig. 4) but allows one cycle of ATP hydrolysis to occur21

10

resulting in the semi-open ADP-bound state (D). Upon dissociation of ADP, state A is formed

11

again which is likely to bind two ATP molecules considering the millimolar concentration of

12

ATP in the cell thus yielding the locked B state regardless of the presence of MalE/maltose.

13

Likewise, initial binding of EIIAGlc to the ADP-bound conformation (III) ultimately results in

14

state B (through D and A).

15

Inducer exclusion as a means to control carbohydrate utilization is not confined to enteric

16

bacteria but is also operating in bacteria belonging to the phylum Firmicutes (low G+C gram-

17

positive bacteria)58. Here, another component of the PTS, HPr, when phosphorylated at

18

residue S46 (P-Ser-HPr) which is distinct of the phosphorylation site used for glucose

19

transport, inhibits transport systems for Class B substrates, e.g. a maltodextrin ABC

20

transporter of Lactobacillus casei59. The underlying mechanism is currently unknown but the

21

data presented here might be helpful to understand inducer exclusion in these bacteria at the

22

molecular level.

23 21


Biochemistry

Page 22 of 45

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

ACKNOWLEDGEMENTS

2 3

We thank Bettina Müller, Britta Kuhnert, Martin L. Daus, Katharina Thoene, Ulf

4

Bergmann, Constanze Homburg, Heidi Landmesser (all Humboldt University of Berlin),

5

Simon Böhm (ETH Zurich), Tufa Assafa and Alexander Heinisch (Free University of Berlin)

6

for their contributions during the initial stage of the project. E. B. would like to thank G.

7

Jeschke

8

Forschungsgemeinschaft for funding the Bruker AWG E580 Q-band spectrometer (INST

9

130/972-1 FUGG).

(ETH Zurich) for providing

the

Q-band

resonator and

the

Deutsche

10 11

Supporting Information Available

12

A list of strains and plasmids used in this study is included in the associated supplementary

13

information. In addition, Cβ- Cβ distances of residues chosen for cross-linking as determined

14

from the X-ray structure, ATPase activities of all transporter variants in the absence and

15

presence of EIIAGlc variants, an immunoblot analysis of cross-linked products and DEER

16

experiments showing the negligible effects of ghost peaks artefacts are included.

17 18 19 20 21 22 23

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22


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(7) Chen, J. (2013) Molecular mechanism of the Escherichia coli maltose transporter, Curr Opin Struct Biol 23, 492–498.

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Remington, S. J. (1991) Three-dimensional structure of the Escherichia coli phosphocarrier

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10

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Structural studies of the Escherichia coli signal transducing protein IIAGlc: implications for

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target recognition, Biochemistry 36, 16087–16096.

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phosphocysteine form of the IIBGlc domain and its binding interface with the IIAGlc subunit,

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protein IIAGlucose and the cytoplasmic domain of the glucose transporter IICBGlucose of the

20

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gene regulation, inducer exclusion, and subunit assembly, J Biol Chem 277, 3708–3717.

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278, 35265–35271.

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surface of the signal transducing protein EIIAGlc that interacts with the MalK subunits of the

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maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium, J Biol

15

Chem 281, 12833-12840.

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(29) Sondej, M., Seok, Y.-J., Badawi, P., Koo, B.-M., Nam, T.-W., and Peterkofsky, A.

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(2000) Topography of the surface of the Escherichia coli phosphotransferase system protein

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enzyme IIAGlc that interacts with lactose permease, Biochemistry 39, 2931–2939.

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(30) Wang, A. Peterkofsky, G.S., and Clore, G. M. (2000) A novel membrane anchor

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function for the N-terminal amphipathic sequence of the signaltransducing protein IIAGlucose

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of the Escherichia coli phosphotransferase system, J Biol Chem 275, 39811–39814.

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(31) Wang, G.S., Keifer, P., and Peterkofsky, A. (2003) Solution structure of the N-

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terminal amphitropic domain of Escherichia coli glucose-specific enzyme IIA in membrane-

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mimetic micelles. Protein Sci 12, 1087–1096.

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(32) Böhm, S., Licht, A., Wuttge, S., Schneider, E., Bordignon, E. (2013) Conformational plasticity of the type I maltose ABC importer, Proc Natl Acad Sci (USA) 110, 5492-5497.

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(33) Orelle, C. Ayvaz, T. Everly, R.M. Klug, C.S., and Davidson, A.L. (2008) Both

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maltose-binding protein and ATP are required for nucleotide-binding domain closure in the

8

intact maltose ABC transporter, Proc Natl Acad Sci (USA) 105, 12837–12842.

9

(34) Hunke, S., Mourez, M., Jéhanno, M., Dassa, E., and Schneider, E. (2000) ATP

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modulates subunit-subunit interactions in an ATP-binding-cassette transporter (MalFGK2)

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determined by site-directed chemical cross-linking, J Biol Chem 275, 15526-15534.

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(35) Miller, J.H. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (36) Moore, J.T., Uppal, A., Maley, F., and Maley G.F. (1993) Overcoming inclusion body formation in a high-level expression system, Protein Expression Purif 4, 160-163.

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(37) Daus, M.L., Landmesser, H., Schlosser, A., Müller, P., Herrmann, A., and Schneider,

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E. (2006) ATP induces conformational changes of periplasmic loop regions of the maltose

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ATP-binding cassette transporter, J Biol Chem 281, 3856-3865.

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(38) Grote, M., Polyhach, Y., Jeschke, G., Steinhoff, H.-J., Schneider, E., and Bordignon,

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E. (2009) Transmembrane signaling in the maltose ABC transporter MalFGK2-E. Periplasmic

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MalF-P2 loop communicates substrate availability to the ATP-bound MalK dimer, J Biol

22

Chem 284, 17521-17526.

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(39) Daus, M.L., Berendt, S., Wuttge, S., and Schneider, E. (2007) Maltose binding protein

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(MalE) interacts with periplasmic loops P2 and P1, respectively, of the MalFG subunits of the

3

maltose ATP-binding cassette transporter (MalFGK2) from Escherichia coli/Salmonella

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during the transport cycle, Mol Microbiol 66, 1107-1122.

5

(40) Ritchie, T.K., Grinkova, Y.V., Bayburt, T.H., Denisov, I.G., Zolnerciks, J.K., Atkins,

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W.M., and Sligar, S.G. (2008) Reconstitution of membrane proteins in phospholipid bilayer

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nanodiscs, Methods Enzymol 464 211–232.

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(41) Daus, M.L., Grote, M., Müller, P., Doebber, M., Herrmann, A., Steinhoff, H.-J.,

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Dassa, E., and Schneider, E. (2007) ATP-driven MalK dimer closure and re-opening and

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conformational changes of the ‘EAA’ motifs are crucial for function of the maltose ATP-

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binding cassette transporter (MalFGK2), J Biol Chem 282, 22387-22396.

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(42) Loo, T.W., and Clarke, D.M. (2001) Determining the dimensions of the drug-binding

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domain of human P-glycoprotein using thiol cross-linking compounds as molecular rulers, J

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Biol Chem 276, 36877-36880.

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(43) Polyhach, Y., Bordignon, E., Tschaggelar, R., Gandra, S., Godt, A., and Jeschke, G.

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(2012) High sensitivity and versatility of the DEER experiment on nitroxide radical pairs at

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Q-band frequencies, Phys Chem Chem Phys 14, 10762-10773.

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(44) Jeschke, G., Chechik, V., Ionita, P., Godt, A., Zimmermann, H., Banham, J., Timmel,

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C.R., Hilger, D., and Jung, H. (2006). DeerAnalysis2006—a comprehensive software package

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for analyzing pulsed ELDOR data. Appl Magn Reson 30, 473–498.

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(45) Kobashi, K. (1968) Catalytic oxidation of sulfhydryl groups by o-phenanthroline copper complex, Biochim Biophys Acta 158, 239-245.

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(46) Falke, J.J., and Koshland, D.E. (1987) Global flexibility in a sensory receptor: A sitedirected cross-linking approach, Science 237, 1596-1600.

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(47) Miller, S., Edwards, M.D., Ozdemir, C., and Booth, I.R. (2003) The closed structure of

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the MscS mechanosensitive channel: Cross-linking of single cysteine mutants, J Biol Chem

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278, 32246-32250.

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(48) Oldham, M. L., Khare, D., Quiocho, F.A., Davidson, A.L., and Chen, J. (2007) Crystal structure of a catalytic intermediate of the maltose transporter, Nature 450, 515-522.

8

(49) Lu, G., Westbrooks, J.M., Davidson, A.L., and Chen, J. (2005) ATP hydrolysis is

9

required to reset the ATP-binding cassette dimer into the resting-state conformation, Proc

10 11 12 13 14

Natl Acad Sci (USA) 102, 17969-17974. (50) Khare, D., Oldham, M.L., Orelle, C., Davidson, A.L., and Chen, J. (2009) Alternating access in maltose transporter mediated by rigid-body rotations, Mol Cell 33, 528-536. (51) Oldham, M.L., and Chen, J. (2011) Crystal structure of the maltose transporter in a pretranslocation intermediate state, Science 332, 1202-1205.

15

(52) Oloo, E.O., Fung, E.Y., and Tieleman, D.P. (2006) The dynamics of the MgATP-

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driven closure of MalK, the energy-transducing subunit of the maltose ABC transporter, J

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Biol Chem 281, 28397–28407.

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(53) Richet, E., Davidson, A.L., and Joly, N. (2012) The ABC transporter MalFGK2

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sequesters the MalT transcription factor at the membrane in the absence of cognate substrate,

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Mol Microbiol 85, 632–647.

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(54) Bakker, E.P., and Randall, L.L. (1984) The requirement for energy during export of b-

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lactamase in Escherichia coli is fulfilled by the total protonmotive force, EMBO J 3, 895–

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(55) Bennett, B.D., Kimball, E.H., Gao, M., Osterhout, R., Van Dien, S.J., and Rabinowitz,

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J.D. (2009) Absolute metabolite concentrations and implied enzyme active site occupancy in

3

Escherichia coli, Nat Chem Biol. 5, 593-599.

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(56) Yaginuma, H., Kawai, S., Tabata, K.V., Tomiyama, K., Kakizuka, A., Komatsuzaki,

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T., Noji, H., and Imamura, H. (2014) Diversity in ATP concentrations in a single bacterial cell

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population revealed by quantitative single-cell imaging, Sci Rep 4, 6522.

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(57) Davidson, A.L., Shuman, H.A., and Nikaido, H. (1992) Mechanism of maltose

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transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins, Proc

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(58) Deutscher, J., Aké, F.M., Derkaoui, M., Zébré, A.C., Cao, T.N., Bouraoui, H.,

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Kentache, T., Mokhtari, A., Milohanic, E., and Joyet, P. (2014) The bacterial

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phosphoenolpyruvate:carbohydrate

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phosphorylation and phosphorylation-dependent protein-protein interactions, Microbiol Mol

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Biol Rev. 78, 231-256.

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phosphotransferase

system: regulation

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(59) Monedero, V. Yebra, M.J. Poncet, S. Deutscher, J. (2008) Maltose transport in Lactobacillus casei and its regulation by inducer exclusion. Res Microbiol 159, 94-102.

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Biochemistry

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

FIGURE LEGENDS

2 3

Fig. 1. Overview of mutants and setup of cross-linking experiments. Structures of the MalK

4

dimer (PDB ID: 4JBW) (A), EIIAGlc (PDB ID: 2F3G) (B), MalFGK2 complexed with EIIAGlc

5

(PDB ID: 4JBW) (C), and experimental setup (D). The figures were drawn with Biovia

6

Discovery Studio Visualizer v4.5 (Dassault Systèmes). A. Residues from the N-terminal

7

subdomain of MalK-A and the regulatory (C-terminal) subdomain of MalK-B considered for

8

cross-linking to EIIAGlc are shown in space-fill representation and coloured green and red,

9

respectively. Residues V17 and E128’ used for demonstrating ATP- and MalE/maltose-

10

induced closing of the MalK dimer by EPR are shown in ball and stick representation and

11

coloured purple. B. Residues of EIIAGlc from binding sites I and II28 considered for cross-

12

linking to MalK2 are shown in space-fill representation and coloured yellow. Residue E160

13

located opposite to the contact sites and used as control is also shown. Residue F88 used as

14

control (when mutated to Q) in EPR experiments is also indicated. C. The MalK monomers

15

are shown in dark grey (MalK-A) and light grey (MalK-B) while both EIIAGlc molecules are

16

shown in blue and cyan, respectively. Residues considered for cross-linking MalK and

17

EIIAGlc are indicated and presented in space-fill representation. Colour code: green, residues

18

from the N-terminal subdomain of MalK-A; red, residues from the C-terminal subdomain of

19

MalK-B; yellow, residues from EIIAGlc. V17 and E128` from MalK-A and MalK-B,

20

respectively, used for DEER experiments, are shown in purple and in ball and stick

21

representation. MalF and MalG are shown in light and dark grey, respectively. D. Cartoon

22

illustrating the experimental setup of the cross-linking experiments. Mono-cys variants of

23

MalF*G*K(C40S)2 are incorporated into MalE/maltose loaded proteoliposomes in two 31


Biochemistry

Page 32 of 45

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

possible orientations. Only complex molecules exposing the MalK subunits to the medium

2

side have contact to mono-cys variants of EIIAGlc and are accessible to cross-linking reagents.

3 4

Fig. 2. Cross-linking of mono-cys variants of the N-terminal subdomain of MalK with EIIAGlc

5

variants. A. Proteoliposomes containing MalF*G*K(C40S, E119C)2 (31 µg) and loaded with

6

MalE/maltose were incubated with EIIAGlc(K69C) (10 µg) or EIIAGlc∆1-16(K69C), the latter

7

lacking the N-terminal sixteen amino acids, in the presence of CuPhe (3 mM CuSO4 and 9

8

mM 1,10-phenanthroline) and cofactors (4 mM ATP/0.1 mM EDTA; 4 mM ATP/4 mM

9

MgCl2; 4 mM ADP) for 20 min at room temperature. Subsequently, the reactions were

10

terminated by adding 5 mM NEM and analysed by SDS-PAGE. B. Cross-linking of

11

MalF*G*K(C40S, K119C) with EIIAGlc(K69C). Reactions were started by adding CuPhe to

12

proteoliposomes and EIIAGlc (left panel: 10 µg; right panel: 2 µg) in 50 mM Tris-HCl, pH

13

7.5. After 10 s at room temperature reactions were terminated by adding 5 mM NEM and

14

aliquots were analyzed by SDS-PAGE. C. Cross-linking of MalF*G*(C40S, K119C) with

15

EIIAGlc(E160C). Reactions were started by adding CuPhe (3 mM CuSO4 and 9 mM 1,10-

16

phenanthroline) or thiosulfonate linkers (EBS, HBS, PBS) at 1 mM final concentration from

17

freshly prepared stock solutions (100 mM in dimethylsulfoxide) to the indicated

18

proteoliposomes (containing 31 µg of complex protein) and EIIAGlc (10 µg) in 50 mM Tris-

19

HCl, pH 7.5, 10% glycerol, 150mM NaCl. D. Cross-linking of MalF*G*K(C40S, Q122C)2

20

with indicated EIIAGlc variants and in the presence of CuPhe and cofactors as described under

21

(A).

22

32


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Biochemistry

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Fig. 3. Cross-linking of mono-cys variants of the C-terminal subdomain of MalK with EIIAGlc

2

variants. A. Proteoliposomes containing MalF*G*K(C40S, A320C)2 were incubated with

3

EIIAGlc(P125C) or EIIAGlc∆1-16(P125C), the latter lacking the N-terminal sixteen amino

4

acids, in the presence of CuPhe and cofactors (4 mM ATP/0.1 mM EDTA; 4 mM ATP/4 mM

5

MgCl2; 4 mM ADP). Protein samples were analyzed by SDS-PAGE. B. Proteoliposomes

6

containing MalF*G*K(C40S, R322C)2 were incubated with EIIAGlc(P125C) or EIIAGlc∆1-

7

16(P125C) and analysed as in (A). See legend to Fig. 2 for further details.

8 9

Fig. 4. DEER analysis of spin-labeled pairs of MalE-FGK2 in nanodiscs. Q-band DEER

10

primary data [V(t)/V(0)] with fitted background (left), background-corrected DEER traces

11

[F(t)/F(0)] with fitted distribution function (center) and corresponding distance distribution

12

(right) calculated using DeerAnalysis2015 for the spin-labeled pair 17MalK/128MalK. A.

13

Samples in the absence of EIIAGlc. Traces are shown in the absence of nucleotides (apo,

14

cyan), after incubation with ATP and MgCl2 (red) and ATP-EDTA (dark green). B.

15

Analogous data in the presence of EIIAGlc. Color code as in panel A. C. In light green, the

16

analysis of a sample previously incubated with ATP and EDTA and afterwards incubated

17

with EIIAGlc. In brown, sample incubated with EIIAGlc(F88Q) and ATP-EDTA. DEER

18

experiments showing the negligible effects of ghost peaks artefacts are shown in Fig. S2.

19 20

Fig. 5. Schematic model of inhibition of the maltose transporter by EIIAGlc. In the absence of

21

EIIAGlc, the transporter switches from an inward facing (open MalK dimer) (I) to an outward

22

facing conformation (closed MalK dimer) upon binding of ATP and maltose-loaded MalE

23

(II). Maltose is released into the translocation path and, following ATP hydrolysis, delivered 33


Biochemistry

Page 34 of 45

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

to the cytoplasm (III). After dissociation of ADP which keeps the MalK dimer in a semi-open

2

state (III) the transporter returns to the inward-facing conformation. EIIAGlc interacts with the

3

transporter residing in any of these conformations (A-D, red arrows) but once the transporter

4

switches to the inward-facing conformation it locks the MalK dimer in the open state, thereby

5

blocking the cycle. Red boxes highlight the blocked (B) and transient states (A, D) by solid

6

and dashed lines, respectively. See text for further details. RD, regulatory (sub)domain.

7 8

Fig. 1

9

10

34


Page 35 of 45

Biochemistry

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

35


Biochemistry

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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Fig. 2

2 3 4

36


Page 37 of 45

Biochemistry

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Fig. 3

2 3 4 5 6 7 8 37


Biochemistry

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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Fig. 4

2

3

38


Page 39 of 45

Biochemistry

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Fig. 5

2

3 4 5 6

39


Biochemistry

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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

For Table of Contents Use Only

2

Mode of interaction of the signal-transducing protein EIIAGlc with the maltose ABC

3

transporter in the process of inducer exclusion

4

Steven Wuttge1§$, Anke Licht1§, M. Hadi Timachi2&, Enrica Bordignon2&*, and Erwin

5

Schneider1*

6

7

40


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Overview of mutants and setup of cross-linking experiments. Structures of the MalK dimer (PDB ID: 4JBW) (A), EIIAGlc (PDB ID: 2F3G) (B), MalFGK2 complexed with EIIAGlc (PDB ID: 4JBW) (C), and experimental setup (D). The figures were drawn with Biovia Discovery Studio Visualizer v4.5 (Dassault Systèmes). A. Residues from the N-terminal subdomain of MalK-A and the regulatory (C-terminal) subdomain of MalK-B considered for cross-linking to EIIAGlc are shown in space-fill representation and coloured green and red, respectively. Residues V17 and E128’ used for demonstrating ATP- and MalE/maltose-induced closing of the MalK dimer by EPR are shown in ball and stick representation and coloured purple. B. Residues of EIIAGlc from binding sites I and II28 considered for cross-linking to MalK2 are shown in space-fill representation and coloured yellow. Residue E160 located opposite to the contact sites and used as control is also shown. Residue F88 used as control (when mutated to Q) in EPR experiments is also indicated. C. The MalK monomers are shown in dark grey (MalK-A) and light grey (MalK-B) while both EIIAGlc molecules are shown in blue and cyan, respectively. Residues considered for cross-linking MalK and EIIAGlc are indicated and presented in space-fill representation. Colour code: green, residues from the N-terminal subdomain of MalKA; red, residues from the C-terminal subdomain of MalK-B; yellow, residues from EIIAGlc. V17 and E128` from MalK-A and MalK-B, respectively, used for DEER experiments, are shown in purple and in ball and stick representation. MalF and MalG are shown in light and dark grey, respectively. D. Cartoon illustrating the experimental setup of the cross-linking experiments. Mono-cys variants of MalF*G*K(C40S)2 are incorporated into MalE/maltose loaded proteoliposomes in two possible orientations. Only complex molecules exposing the MalK subunits to the medium side have contact to mono-cys variants of EIIAGlc and are accessible to cross-linking reagents. 177x155mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cross-linking of mono-cys variants of the N-terminal subdomain of MalK with EIIAGlc variants. A. Proteoliposomes containing MalF*G*K(C40S, E119C)2 (31 µg) and loaded with MalE/maltose were incubated with EIIAGlc(K69C) (10 µg) or EIIAGlc∆1-16(K69C), the latter lacking the N-terminal sixteen amino acids, in the presence of CuPhe (3 mM CuSO4 and 9 mM 1,10-phenanthroline) and cofactors (4 mM ATP/0.1 mM EDTA; 4 mM ATP/4 mM MgCl2; 4 mM ADP) for 20 min at room temperature. Subsequently, the reactions were terminated by adding 5 mM NEM and analysed by SDS-PAGE. B. Cross-linking of MalF*G*K(C40S, K119C) with EIIAGlc(K69C). Reactions were started by adding CuPhe to proteoliposomes and EIIAGlc (left panel: 10 µg; right panel: 2 µg) in 50 mM Tris-HCl, pH 7.5. After 10 s at room temperature reactions were terminated by adding 5 mM NEM and aliquots were analyzed by SDS-PAGE. C. Cross-linking of MalF*G*(C40S, K119C) with EIIAGlc(E160C). Reactions were started by adding CuPhe (3 mM CuSO4 and 9 mM 1,10-phenanthroline) or thiosulfonate linkers (EBS, HBS, PBS) at 1 mM final concentration from freshly prepared stock solutions (100 mM in dimethylsulfoxide) to the indicated proteoliposomes (containing 31 µg of complex protein) and EIIAGlc (10 µg) in 50 mM Tris-HCl, pH 7.5, 10% glycerol, 150mM NaCl. D. Crosslinking of MalF*G*K(C40S, Q122C)2 with indicated EIIAGlc variants and in the presence of CuPhe and cofactors as described under (A). 177x146mm (300 x 300 DPI)


Page 42 of 45

Page 43 of 45

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Biochemistry

Cross-linking of mono-cys variants of the C-terminal subdomain of MalK with EIIAGlc variants. A. Proteoliposomes containing MalF*G*K(C40S, A320C)2 were incubated with EIIAGlc(P125C) or EIIAGlc∆116(P125C), the latter lacking the N-terminal sixteen amino acids, in the presence of CuPhe and cofactors (4 mM ATP/0.1 mM EDTA; 4 mM ATP/4 mM MgCl2; 4 mM ADP). Protein samples were analyzed by SDS-PAGE. B. Proteoliposomes containing MalF*G*K(C40S, R322C)2 were incubated with EIIAGlc(P125C) or EIIAGlc∆116(P125C) and analysed as in (A). See legend to Fig. 2 for further details. 85x157mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

DEER analysis of spin-labeled pairs of MalE-FGK2 in nanodiscs. Q-band DEER primary data [V(t)/V(0)] with fitted background (left), background-corrected DEER traces [F(t)/F(0)] with fitted distribution function (center) and corresponding distance distribution (right) calculated using DeerAnalysis2015 for the spinlabeled pair 17MalK/128MalK. A. Samples in the absence of EIIAGlc. Traces are shown in the absence of nucleotides (apo, cyan), after incubation with ATP and MgCl2 (red) and ATP-EDTA (dark green). B. Analogous data in the presence of EIIAGlc. Color code as in panel A. C. In light green, the analysis of a sample previously incubated with ATP and EDTA and afterwards incubated with EIIAGlc. In brown, sample incubated with EIIAGlc(F88Q) and ATP-EDTA. 159x117mm (300 x 300 DPI)


Page 44 of 45

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Biochemistry

Schematic model of inhibition of the maltose transporter by EIIAGlc. In the absence of EIIAGlc, the transporter switches from an inward facing (open MalK dimer) (I) to an outward facing conformation (closed MalK dimer) upon binding of ATP and maltose-loaded MalE (II). Maltose is released into the translocation path and, following ATP hydrolysis, delivered to the cytoplasm (III). After dissociation of ADP which keeps the MalK dimer in a semi-open state (III) the transporter returns to the inward-facing conformation. EIIAGlc interacts with the transporter residing in any of these conformations (A-D, red arrows) but once the transporter switches to the inward-facing conformation it locks the MalK dimer in the open state, thereby blocking the cycle. Red boxes highlight the blocked (B) and transient states (A, D) by solid and dashed lines, respectively. See text for further details. RD, regulatory (sub)domain. 177x122mm (300 x 300 DPI)


Mode of Interaction of the Signal-Transducing Protein EIIAGlc with the

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