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Crystal Structure of Maik, the Atpase Subunit of the Trehalose/Maltose

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Crystal Structure of Maik, the Atpase Subunit of the Trehalose/Maltose The EMBO Journal Vol. 19 No. 22 pp. 5951±5961, 2000 Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis Kay Diederichs, Joachim Diez, ®brosis transmembrane conductance regulator (CFTR) Gerhard Greller, Christian MuÈ ller1, involved in the inherited disease cystic ®brosis, sterol Jason Breed2, Christoph Schnell, transporters, eye pigment precursor importers and protein Clemens Vonrhein3, Winfried Boos and exporters. Even though the arrayof substrates seems Wolfram Welte4 endless and the molecular architecture can be rather diverse, the ATPase module is an essential and conserved Fachbereich Biologie, UniversitaÈt Konstanz, M656, D-78457 Konstanz, subunit of all transporters. Its sequence features are the 1 Germany, School of Bioscience, Cardiff University, 10 Museums main basis for the identi®cation of new familymembers Avenue, PO Box 911, Cardiff CF10 3US, 2Astra Zeneca, Mereside, Maccles®eld SK10 4TG and 3Global Phasing Ltd, Sheraton House, (Holland and Blight, 1999). Castle Park, Cambridge CB3 0AX, UK A subfamilyof ABC transporters are binding protein- dependent systems that are ubiquitous in eubacteria and 4Corresponding author e-mail: [email protected] archaea, where theycatalysethe high-af®nityuptake of small polar substrates into the cell. One of the best studied The members of the ABC transporter family trans- examples is the E.coli maltose±maltodextrin system (Boos port a wide variety of molecules into or out of cells and Lucht, 1996; Boos and Shuman, 1998). It consists of a and cellular compartments. Apart from a trans- binding protein (MalE) as its major substrate recognition location pore, each member possesses two similar site, located in the periplasm. Two homologous hydro- nucleoside triphosphate-binding subunits or domains phobic membrane proteins (MalF and MalG) form a in order to couple the energy-providing reaction with heterodimeric translocation pore with a dimer of the ATP- transport. In the maltose transporter of several hydrolysing subunit (MalK) associated from the cytoplas- Gram-negative bacteria and the archaeon Thermo- mic side. Formation of the MalEFGK2 transport complex coccus litoralis, the nucleoside triphosphate-binding therefore couples ATP hydrolysis with active transport of subunit contains a C-terminal regulatory domain. A substrate. dimer of the subunit is attached cytoplasmically to the The E.coli MalK (E.c.MalK) and its Salmonella translocation pore. Here we report the crystal struc- typhimurium homologue (S.t.MalK), which share 95% ture of this dimer showing two bound pyrophosphate identical residues, have been subject to intense analysis molecules at 1.9 AÊ resolution. The dimer forms by ever since their discovery(Bavoil et al., 1980; Shuman association of the ATPase domains, with the two regu- and Silhavy, 1981). Studies of enzymatic activity as an latory domains attached at opposite poles. Signi®cant ATPase (Morbach et al., 1993; Davidson et al., 1996) have deviation from 2-fold symmetry is seen at the interface been performed. The homodimeric subunit interaction has of the dimer and in the regions corresponding to those been analysed (Davidson and Sharma, 1997; Kennedy and residues known to be in contact with the translocation Traxler, 1999), and the requirements for its assemblyin pore. The structure and its relationship to function the transport complex (Davidson and Nikaido, 1991; are discussed in the light of known mutations from Panagiotidis et al., 1993; Lippincott and Traxler, 1997) the homologous Escherichia coli and Salmonella have been recognized. Mutational analysis for domain typhimurium proteins. interactions with its cognate membrane components Keywords: active transport/CFTR/crystal structure/ (Mourez et al., 1997) as well as cross-linking studies maltose uptake and regulation/P-glycoprotein (Hunke et al., 2000a) have been reported. Mutational analysis has also de®ned the functional importance of conserved regions such as Walker-A, Walker-B and the switch region for ATP binding, as well as the signature Introduction motif region and the helical domain for coupling of ATP ABC transporters are found in all eubacterial, archaeal and hydrolysis to transport (for a review see Schneider and eukaryotic species studied to date and most probably Hunke, 1998), and has revealed a remarkable versatilityof represent the largest familyof homologous proteins. In MalK to interact with different regulatoryproteins. Escherichia coli, an estimated 5% of the whole genome According to other studies, MalK is able to interact with encodes them (Linton and Higgins, 1998). unphosphorylated EIIAGlc, a subunit of the phosphotrans- ABC transporters are modular mechanical machines ferase (PTS)-type glucose transporter, leading to the that couple ATP hydrolysis to the physical movement of inhibition of maltose transport (Dean et al., 1990; molecules through membranes. Several subclasses can be Vandervlag and Postma, 1995), a phenomenon called de®ned according to the direction of substrate trans- inducer exclusion. In addition, the C-terminus of MalK is location, substrate speci®cityand subunit organization. able to affect mal gene regulation (KuÈhnau et al., 1991) by Prominent familymembers are P-glycoprotein involved in interacting with and inactivating MalT, the speci®c gene multiple drug resistance, the gated chloride channel cystic activator of mal gene expression (Panagiotidis et al., ã European Molecular Biology Organization 5951 K.Diederichs et al. Table I. Statistics on data reduction and MAD phasing for MalK data sets Native HgCl2 peak in¯ection remote high remote low Wavelength (AÊ ) 0.8424 1.0080 1.0089 1.0163 1.0000 Resolutiona (AÊ ) 1.86 (1.88±1.86) 2.65 (2.68±2.65) 2.65 (2.68±2.65) 2.65 (2.68±2.65) 2.65 (2.68±2.65) Redundancya 4.6 (3.0) 4.0 (4.0) 4.0 (4.1) 4.0 (3.2) 4.0 (4.1) No. of unique observationsa 82 668 (2430) 29 141 (963) 29 172 (965) 29 158 (934) 29 183 (963) Completeness (%) 99.0 (93.8) 99.0 (99.8) 99.1 (100.0) 99.0 (96.8) 99.1 (99.8) R-measb 3.9 (28.0) 4.2 (13.9) 4.1 (16.3) 4.0 (17.5) 4.0 (15.8) R-mrgd-Fb 4.1 (27.2) 3.7 (10.8) 3.8 (12.9) 4.0 (15.8) 3.8 (12.2) Isomorphous phasing ± ± 5.61 6.45 3.41 power (acentric re¯ections) Anomalous phasing ± 1.88 1.23 1.13 0.25 power (acentric re¯ections) Figure of merit ± 0.69 aThe values for the highest resolution shell are given in parentheses. bR-mrgd-F as de®ned byDiederichs and Karplus (1997). 1998), demonstrating a link between transport of substrate more easily. The recombinant protein was puri®ed from and gene regulation. the soluble cellular extract of strain BL21, which lacks Binding protein-dependent ABC transporters have also several proteases (Studier and Moffatt, 1986), as described been found in thermophilic bacteria (Herrmann et al., previously. The ATPase activity corresponded to that of 1996; Sahm et al., 1996). Recently, we described an ABC the previouslypublished construct containing a C-terminal transporter for maltose/trehalose in the hyperthermophilic His tag. archaeon Thermococcus litoralis (Xavier et al., 1996). The crystals comprise two molecules (termed A and B) This transport system has several unusual properties: it per asymmetric unit, and the structure was solved by shows a high af®nity( Km of ~20 nM) at 85°C, the multiwavelength anomalous diffraction (MAD) analysis of optimum growth temperature of this organism, and it an HgCl2 derivative. As onlyone atom of Hg per molecule recognizes its verydifferent substrates, maltose and of MalK was bound, the phasing power was low. trehalose, with equal af®nitybut does not bind larger Furthermore, despite cryogenic conditions, the isomor- maltodextrins. Its cognate binding protein has been phous and anomalous signal stronglydecreased with puri®ed (Horlacher et al., 1998) and its crystal structure exposure time, presumablydue to radiation damage. The has been solved (J.Diez, K.Diederichs, G.Greller, crystallographic analysis therefore ®rst employed single R.Horlacher, W.Boos and W.Welte, manuscript submit- anomalous diffraction (SAD) phasing at the peak wave- ted). The T.litoralis MalK (T.l.MalK) has been hetero- length, and was then extended to include all four wave- logouslyexpressed in E.coli and its biochemical properties lengths. To stabilize the heavyatom re®nement process have been studied. Its sequence, size (372 residues) and in the four-wavelength case, the Hendrickson± biochemical properties reveal its close relationship to the Lattman coef®cients of the SAD phases were introduced E.c.MalK protein. It optimally hydrolyses ATP at 85°C as external restraints. The phasing power values given by and exhibits a Km of 150 mM for ATP at this temperature SHARP (de la Fortelle and Bricogne, 1997; Table I) are (Greller et al., 1999). much higher for the isomorphous than for the anomalous Little is known about how ATP hydrolysis, presumably signal, which is atypical for a MAD experiment and is via a series of protein conformational changes (Ehrmann more reminiscent of a multiple isomorphous replacement et al., 1998), is coupled to the mechanism of transport. (MIR) analysis. Thus, structural information about the translocating com- After initial automatic ARP/wARP (Lamzin and plex as well as the ATP-coupling structures is needed. As to Wilson, 1993) model building, model re®nement was the subclass of importers, the onlyknown atomic structure continued using standard procedures at 1.9 AÊ . is that of HisP, the ATP-hydrolysing subunit of the histidine Although T.l.MalK was crystallized in the presence of transporter of S.typhimurium (Hung et al., 1998). ADP, there was onlyclear densityfor a pyrophosphate Here we present the crystal structure of T.l.MalK, the molecule. At the expected position of the adenosine group, energy-coupling subunit of the trehalose/maltose trans- the map shows elongated densitythat could result from porter of T.litoralis, at 1.9 AÊ resolution.
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