Conformational dynamics in binding-protein-independent mutant of

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Conformational dynamics in binding-protein-independent mutant of the Escherichia coli maltose transporter, MalG511 and its interaction with maltose binding protein Ruchika Bajaj, Mariana I. Park, Cynthia V. Stauffacher, and Amy L. Davidson Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00266 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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Biochemistry

Conformational dynamics in binding-protein-independent mutant of the Escherichia coli maltose transporter, MalG511 and its interaction with maltose binding protein

Ruchika Bajaj†*, Mariana I. Park§, Cynthia V. Stauffacher† and Amy L. Davidson§

†Hockmeyer Hall of Structural Biology, Department of Biological Sciences, Purdue University § Department of Chemistry, Purdue University, West Lafayette, Indiana – 47906

Running title: Catalytic cycle of MalG511 Keywords: Binding-protein-independent mutant, maltose transporter, electron paramagnetic resonance spectroscopy, ATPase assay

‡ Dedicated to the memory of Professor Amy L. Davidson, Purdue University

*Corresponding Author Ruchika Bajaj, Ph.D. University of California San Francisco Mission Bay Campus, Rock Hall 581 1550 4th Street, San Francisco, CA 94143-2911 [email protected] Phone number: +1 (415) 530-0481

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ABBREVIATIONS: MalFGK2, maltose transporter; MBP, maltose binding protein; ATP, adenosine tri-phosphate; ABC, ATP binding cassette; BPI, binding-protein-independent; MalG511, binding-protein-independent mutant of Escherichia coli maltose transporter; MSP, membrane scaffolding protein; SDSL, spin directed spin labeling; EPR, electron paramagnetic resonance


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Biochemistry

ABSTRACT MalG511 is a genetically selected binding-protein-independent mutant of Escherichia coli maltose transporter (MalFGK2), which retains specificity for maltose and shows a high basal ATPase activity in the absence of maltose binding protein (MBP). It shows an intriguing biphasic behavior in maltose transport assays in the presence of MBP, with low levels of MBP stimulating the activity while higher levels (>50µM) inhibit transport activity. Remarkably, the rescuing effect of MBP suppressor mutant, MBPG13D, turns it into an attractive model to study regulatory mechanisms in the ABC transporter superfamily. It is hypothesized that the special characteristics of MalG511 result from mutations that shift its equilibrium towards the transition state of MalFGK2. We tested this hypothesis by using site directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy and showed conformational changes in MalG511 and its interaction with MBP and MBPG13D during its catalytic cycle. We found that the MalG511 utilizes the same alternate access mechanism as MalFGK2, including all three open, semi-open and closed states of the MalK dimer, to transport maltose across the membrane. However, the equilibrium of this mutant is shifted towards the semi-open state in its resting state and interacts with MBP with high affinity, providing an explanation for the inhibition of MalG511 by MBP at higher concentrations. In contrast, the mutant binding protein, MBPG13D, interacts with lower affinity and could restore MalG511 to a normal catalytic cycle.


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INTRODUCTION The ATP binding cassette (ABC) proteins constitute one of the largest protein superfamilies and are found in organisms ranging from bacteria to humans. They harness energy generated from ATP hydrolysis and utilize it for the import or export of a range of substrates [1, 2]. ABC transporters are composed of two transmembrane domains (TMDs) that form a pathway for moving substrates across the membrane and two cytoplasmic nucleotide binding domains (NBDs) which bind and hydrolyze ATP [3, 4]. Overexpression and genetic defects in ABC proteins have been implicated in a number of diseases like cystic fibrosis, hyperinsulinemia, macular degeneration, and multidrug resistance in cancer [5-7]. Therefore, understanding the mechanism of action and regulation of the ABC proteins with regard to their structural details has clinical significance. The maltose transporter (MalFGK2) from Escherichia coli has served as a representative model to understand the fundamental mechanism of coupling of ATP hydrolysis to substrate translocation in the ABC transporter family [1, 8-10]. MalFGK2 is a binding-protein-dependent importer which is composed of two transmembrane subunits, MalF and MalG, and two cytoplasmic NBDs, MalK2 [11]. A soluble, monomeric maltose binding protein (MBP), which transitions from open to closed conformation by the binding of maltose, [12-14] delivers either maltose or maltodextrins from the periplasm to MalFGK2 and stimulates the ATPase activity of MalK domains [1, 15]. Three crystallographic structures of MalFGK2 have provided "snapshots" of different steps of the catalytic cycle of the transporter [16-19]. The crystal structure of MalFGK2 in the resting state, lacking MBP and nucleotides, shows that the transporter assumes an inward-facing conformation in which the transmembrane maltose binding site is exposed to the cytoplasm. In this state, NBDs are widely separated in the open conformation and so display


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Biochemistry

negligible ATPase activity [17]. Another crystal structure of MalFGK2 in complex with maltosebound MBP in a closed conformation showed a pre-translocation state in which the MalK domains have moved closer together to yield a semi-open conformation [18]. Recent findings by our group suggest that ATP stabilizes this pre-translocation state and promotes the concerted closure of the MalK domains and the opening of the bound MBP [20-23]. The closure of the MalK domains and ATP hydrolysis results in an outward facing conformation which is defined as the transition state [16, 19, 24]. Subsequently, the opening of MBP and the insertion of the third periplasmic loop (P-3, scoop loop) from MalG, allows the maltose to be transferred from MBP to the transmembrane maltose binding site [16, 25]. After ATP hydrolysis and release of Pi and/or ADP, the MalFGK2 returns to its resting state to complete the catalytic cycle which involves the disengagement of the MBP C-terminal lobe [22] and the transfer of maltose to the cytoplasm. These structural and functional studies of the MalFGK2 have shown that MBP plays an important role in its catalytic cycle [16, 20, 26]. To understand the role of MBP and its interaction with MalFGK2 in maltose transport, mutant forms of MalFGK2 were genetically selected for the ability to transport maltose and hydrolyze ATP in the absence of MBP. These mutants were termed as binding-protein-independent (BPI) mutants [27]. These mutations were mapped to the periplasmic region which is involved in the transition between inward and outward conformations [17, 28]. ATPase activities of BPI mutants were far higher than MalFGK2 but were still coupled to maltose transport [10, 28]. All BPI mutants also exhibited higher Km values for maltose transport (2 mM for BPI mutants vs 1 µM for MalFGK2), which suggests that these mutants utilize a low affinity site for maltose binding [10]. We have studied one of the remarkable BPI mutants, MalG511, which contains two point mutations in MalG [L135F and I154S] (Figure 1) [27, 28]. Like other BPI mutants, MalG511


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also shows high basal ATPase activity in reconstituted membranes (70 nmol/min/mg for the MalG511 vs 1 nmol/min/mg for the MalFGK2), which can be further stimulated up to five-fold in the presence of MBP [10, 28, 29]. MalG511 has also been reported to have higher rates for maltose transport in intact cells compared with MalFGK2 (200 pmol/min/109 cells for MalG511 vs 2 pmol/min/109 cells for MalFGK2) [28]. However, it exhibits an unusual biphasic behavior in maltose transport assays; maltose transport activity is stimulated by low concentrations of MBP, but is inhibited by high concentrations (>25 µM) of MBP [29]. Furthermore, since the growth of MalG511 strains was found to be inhibited in the presence of MBP on maltose media, some suppressor variants of MBP, such as MBPG13D (originally designated as MBP632), were isolated [30]. These suppressor MBP mutants were able to restore productive interactions with MalG511, as it occurs in wild type counterparts (MalFGK2 and MBP) [28]. Figure 1: Location of mutations in MalG511 in the resting state of MalFGK2 (PDB ID: 2R6G) (A) front view (B) [17] top view, Color codes: MalF, blue; MalG, yellow; two MalK subunits, red and green. Mutated residues, I154S and L135F, in MalG511 are indicated as magenta spheres and labeled in black. Inset (A): interaction between L135 and the periplasmic P4 loop of MalF in resting and transition states (orange, MalG; cyan, MalF) (Modified from [17]) Figure 1


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Biochemistry

This study is mainly designed to address three main questions: first, to elucidate the conformational dynamics in MalG511 during its catalytic cycle in the absence of MBP; second, to identify the underlying cause of unusual biphasic behavior of MalG511 in the presence of MBP and finally, to understand the restoration of its interaction with MBPG13D as in wild type counterparts. Biochemical studies proposed that the BPI mutations in MalG511 shift the equilibrium state of the transporter towards the transition state in the catalytic cycle, which leads to reduction in the activation barrier and higher basal activity [10]. Genetic and structural studies supported this hypothesis by showing that these mutations in BPI mutants are clustered in specific regions of MalF and MalG, primarily at the subunit interaction sites that undergo conformational changes during the inward/outward transition [17, 28]. For instance, in the resting state of MalFGK2,


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the residue L135 in MalG is in close contact with the MalF P4 loop (periplasmic loop 4). This interaction is disrupted in the transition state when the MalG helices rotate during the transition [17] (Figure 1 inset). In contrast, in MalG511, mutation of L135 to phenylalanine destabilizes these interactions due to its bulky ring structure, which shifts the equilibrium to a conformation that more closely resembles the transition state of MalFGK2 [17]. Since MBP is proposed to have more affinity towards the transition state of MalFGK2, MBP may bind MalG511 more strongly, explaining its inhibitory effect at higher concentrations [10, 29]. The rescue effect by the MBP suppressor mutant, MBPG13D, is proposed to result from its low affinity with MalG511 [10, 29]. To test this hypothesis, we have used site directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy to study the opening and closing of MalK domains during the catalytic cycle of MalG511 in the absence or presence of MBP or MBPG13D and the interaction between MBP and MalG511. This study can broaden our understanding of structural regulation in ABC exporters such as P-glycoprotein and cystic fibrosis regulator transporter, which are implicated in multidrug resistance and cystic fibrosis, respectively. As is the case in MalG511, these exporters also exhibit high basal activity and do not utilize any binding protein to transport their substrates [31]. EXPERIMENTAL PROCEDURES Purification and spin labeling of MalG511: In order to co-express the proteins that comprise MalG511, E. coli strain AD126 was transformed with the spectinomycin-resistance plasmid pMS421 [32] that expresses LacIQ, the ampicillin resistance plasmid pLH33 that expresses MalF and mutated MalG with L135F and I154S [10], and the chloramphenicol-resistance plasmid pKJ that expresses MalK with a C-terminal histidine tag [10]. Another strain containing pCO-SSM-


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Biochemistry

V16C-R129C [20] instead of pKJ was constructed in order to express a version of the MalG511 that contains MalK with V16C and R129C substitutions and no native cysteines. MalG511 was overexpressed and purified as described previously [20] except that the equilibration buffer for the Co2+ affinity chromatography was 20 mM Tris pH 8.0 and 150 mM NaCl (Buffer G) with 0.01% n-dodecyl-β-D-maltoside. Spin labeling of the MalG511-V16C/R129C was carried out in the resin bound form during purification. The Co2+-bound protein was incubated with a ten-fold molar excess of 1-oxyl-2,2,5,5-tetramethyl-∆3-pyrroline-3-methyl methanethiosulfonate spin label (MTSL, Toronto Research Chemicals) in a falcon tube overnight with gentle mixing by inversion at 4ºC. The resin was washed excessively with equilibration buffer to remove the free spin label, and the spin-labeled protein was eluted with equilibration buffer containing 100 mM imidazole. The eluted protein sample was concentrated to approximately 70-80 µM and dialyzed overnight at 4ºC. Protein concentration was determined using a Bradford protein assay kit (BioRad, Inc.). Reconstitution of MalG511 in nanodiscs: MalG511 was reconstituted in nanodiscs by using previously described method for MalFGK2 [33]. Membrane scaffolding protein (MSP), MSP1E3D1, was used for nanodiscs preparation. Briefly, MalG511, MSP and lipid (Soybean lipids, Sigma P5638) were mixed together in a specific molar ratio (MalG511: MSP = 1:10, MSP: lipid = 1:50) with cholate at the concentration of 25mM, in a volume of 450 µl. The mixture was kept at room temperature for an hr with gentle rocking, for equilibrium. After that, biobeads SM2 (Bio-Rad) was added to the mixture to remove excess of detergent and trigger self-assembly of nanodiscs and kept at room temperature for next three hours with gentle rocking. Biobeads were removed and sample was centrifuged at 13000 g for 1 min to remove any aggregates.


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Expression and purification of MBP: MBP–BAR1000 was used for overexpression of MBP, MBP-G13D, MBPD41C, and MBP352C. MBP and its mutants were purified as described previously [22, 23]. For spin labeling of the cysteine mutants, 10 mg of mutant MBP was incubated with equimolar concentrations of DTT at room temperature for 30 min. The reduced form of MBP was then mixed with 0.5 ml of Ni-sepharose 6 fast flow resin (GE Healthcare) in a 1 ml solution of Buffer G (20 mM Tris-HCl pH 8.0, 150 mM NaCl) for 30 min at 4ºC. The MBP cysteine mutants bound on resin were then incubated with a 10X molar excess of spin label MTSL overnight with rotation at 4ºC. The resin was loaded on a column, washed extensively with Buffer G to remove any excess of spin label, and then the spin-labeled MBP was eluted with Buffer G containing 100 mM imidazole. The eluted protein sample was concentrated with a centrifugal filter device (Millipore, cut off 10 kD) and dialyzed overnight at 4ºC against Buffer G. The protein concentration was determined spectrophotometrically, using an extinction coefficient of 1.7 (ɛ0.1%1cm) [34]. ATPase activity assay: The ATPase activity of MalG511 in detergent (0.01% n-dodecyl-β-Dmaltoside) or reconstituted in proteoliposomes was measured using an enzyme coupled assay [20]. This assay generates ATP using pyruvate kinase and lactate dehydrogenase with a concomitant decrease in NADH, which can be measured at 340 nm. For a typical reaction, the ATPase mixture contains 50 mM HEPES/KOH pH 8.0, 10 mM MgCl2, 4 mM phosphoenol pyruvate, 60 µg/ml pyruvate kinase, 32 µg/ml lactate dehydrogenase, 0.3 mM NADH and 1.5 mM ATP. Typically, at least 10 µg of transporter was used for each assay and the absorbance was recorded over 8 min. Enzymatic activity was measured with varying concentrations (0-200 µM) of maltose-bound MBP or MBPG13D. The apparent affinity constants for MBP or MBPG13D were determined by analyzing the hyperbolic portion of the curve in these assays.


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Biochemistry

EPR spectroscopy: X-band EPR spectroscopy was carried out on a Bruker EMX-plus with an ER4119HS cavity using Win EPR (v4.40) software. Samples (30 µl) were collected in a capillary tube that was inserted into a quartz tube. Spectra were recorded at room temperature at 10 mW microwave power and 100 kHz modulation amplitude over 1.0 Gauss (G). The spectra were routinely signal-averaged nine times over a 200 G scan width. The amount of spin label incorporated into the protein was determined by comparing the calculated double integral of the signal (Bruker WinEPR Processing software) to a standard curve. The labeling efficiency was determined by calculating the molar ratio of spin label and protein. The raw data obtained through Bruker WinEPR was converted into a MS Excel spreadsheet format through the EMX-file converter (Dr. Michael Everly, Purdue University). The spectra were normalized to either the same number of spins or the same peak heights for comparison. Spin-spin distances were calculated using simulation software Labview Shortdistances106 program written by Dr. Christian Altenbach [35, 36]. This program uses a Fourier deconvolution procedure to prepare a broadening function from spin-spin interactions. This broadening function is fit to the data to obtain a weighted sum of Pake functions corresponding to inter-spin distances. For validation, the experimental spectrum is compared to a simulated spectrum from an appropriate distribution of distances. Only dipolar interactions are considered in the convolution procedures, so the reliability of obtained distance distributions at shorter distances (< 10Å, when exchange coupling is also relevant), is decreased. Various concentrations (70-80 µM) of the MalFGK2, MalG511 or spin-labeled MalG511V16C/R129C were stabilized in detergent micelles or reconstituted in proteoliposomes as described [20]. MBP was added at a concentration of 200 µM. In experiments with spin-labeled MBP, the concentration ratio of MBP:transporter was maintained at 1:2. Samples were incubated


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at room temperature with one or more of the following maltose transporter ligands: 2.5 mM EDTA, 10 mM ATP, 1 mM maltose, and/or 10 mM MgCl2. EDTA concentrations were increased to 12 mM to chelate previously added MgCl2 to revert the transporter to resting state.

RESULTS ATPase activity of the MalG511 exhibits a biphasic behavior with increasing equimolar concentrations of maltose and MBP, which is restored to hyperbolic fashion in the presence of MBPG13D ATPase assays were performed with MalG511 reconstituted in proteoliposomes (Figure 2) as well as in detergent micelles (Figure S1) to confirm its high basal activity in absence of MBP (Figure 2, blue curve). These assays showed that MalG511 had significantly more activity in proteoliposomes than in detergent preparations (550 nmoles/min/mg in proteoliposomes and 225 nmoles/min/mg in detergent). Next, we wanted to see if the unusual biphasic behavior of MalG511 with MBP in maltose transport assays [29] can be mirrored in ATPase assays. Therefore, we examined the effect of increasing equimolar concentrations of maltose and MBP on the ATPase activity of MalG511 in proteoliposomes (Figure 2, red curve). MalG511, in the presence of MBP, follows biphasic behavior in ATPase assays; MBP stimulated the ATPase activity of MalG511 at low concentrations, but showed an inhibitory effect at concentrations above 25 µM. Interestingly, the apparent dissociation constant for the interaction between MalG511 and MBP in the proteoliposomal preparation was calculated to be 2 µM, which is consistent with the predicted high affinity interaction. Furthermore, to understand the role of the MBPG13D suppressor mutant in the catalytic cycle of the MalG511, we also performed ATPase activity assays for


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Biochemistry

MalG511 with increasing equimolar concentrations of MBPG13D and maltose. MBPG13D restored the ATPase activity of the MalG511 at high maltose-MBPG13D concentrations (Figure 2, green curve), in a similar fashion as observed in maltose transport assays [29]. The apparent Kd for the interaction between MalG511 and MBPG13D was 12 µM and 25 µM in detergent and proteoliposomal preparations, respectively, consistent with the low affinity predicted for MBPG13D binding to MalG511 (Figure 2, S1). Figure 2: Basal ATPase activity of MalG511 in proteoliposomes in the absence (♦) or presence of increasing concentrations of maltose-bound MBP (■) or MBPG13D (▲). Data points are from three measurements. Figure 2

MalG511 can undergo conformation changes without requiring MBP or maltose. Site-directed spin labeling (SDSL) and EPR spectroscopy were used to study the relative movements of the spin-labeled MalK (NBD) domains during the catalytic cycle of MalG511. Inter-


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spin distance distributions between two spin labels were calculated using a spectral deconvolution program called “ShortDistances106” as described (Figure S2) and categorized into three classes; large (20-25 Å), intermediate (14-19 Å) and short (8-9 Å) [35, 36]. Spin labeling of cysteine mutants at residues V16 and R129 in opposing MalK subunits was used to monitor the opening and closing of the nucleotide binding interface during the catalytic cycle of the MalFGK2 [20-22]. V16 and R129 are >39Å apart within a single MalK monomer and residue pairs V16/V16’ and R129/R129’ are >35Å apart within the closed MalK dimer (Figure 3). Therefore, any inter-spin signals in doubly spin labeled mutants will result predominantly from the V16/R129' and V16'/R129 interactions [16, 20]. Figure 3: Location of cysteine mutations (V16 and R129) in MalK domains for site directed spin labeling in EPR spectroscopy. MalK domains are in open conformation (PDB ID: 2R6G, Chain A and B, [17]) (A) side view (B) top view. MalK domains are colored in red and green. Residues V16 and R129 in MalK domains are colored in cyan and orange respectively. Figure 3


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Biochemistry

For the EPR experiments, the MalG511 transmembrane domain was expressed with MalK containing either two cysteine residue substitutions at V16 and R129 or only one of these substitutions. The expression levels, yield (5-6 mg/L) and ATPase activity of these cysteine containing mutant transporters were comparable to those of the parental transporter. Both single and double cysteine mutants of the MalG511 were modified with the MTSL spin label at 85% and 70% efficiency, respectively. EPR spectra of doubly spin-labeled MalG511-V16C/R129C in the presence or absence of different ligands was assessed to probe conformational changes in MalG511 during its catalytic cycle. To study different stages in the catalytic cycle, ATP in the presence of EDTA was first added to study the nucleotide-bound form or pre-hydrolysis state of MalG511. Next, Mg2+ was added to promote the post-hydrolysis state of MalG511. Finally, excess EDTA was added to allow the conversion of the MalG511 from the post-hydrolysis state to the resting state [20]. For EPR experiments, detergent as well as proteoliposomal preparations were used in the presence or absence of maltose. The EPR spectrum of the doubly spin-labeled MalG511-V16C/R129C in the resting state consists of two peaks in which the inner mobile component correlates with V16C and the outer immobile component correlates with the more restricted residue R129C (Figure 4). Control experiments with spin-labeled single cysteine mutants (MalG511-V16 and MalG511-R129) showed no or minimal changes in the spectrum upon the addition of the ligands (Figure S3, S4). A comparative analysis of the EPR spectra of the doubly spin-labeled MalG511-V16C/R129C and the spectral sum of each of the singly spin-labeled mutants in proteoliposomes, showed that the distance between the nitroxide spin labels attached to MalK domains for 62% of the population was large (>20Å) in the resting state with no ligands (Figure 4, S5, Table 1). The remaining 38% of the


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population did not interact, which is most likely due to underlabeling at both sites or that both sites are separated beyond the measurable range in this population. This finding is consistent with the MalK domains of the MalG511 existing in an inward-facing open conformation in the resting state [17, 20]. Addition of ATP and EDTA to doubly spin-labeled MalG511V16C/R129C in the absence or presence of maltose showed a little broadening in the spectrum (Figure 4, S5), which could fit to a very broad Gaussian distribution centered at intermediate distances (18-19 Å) (Figure 4, S5, Table 1). When MgCl2 was added to advance MalG511 through ATP hydrolysis in the absence or presence of maltose, there was a significant spectral broadening (Figure 4, S5), which is again consistent with MalK subunits coming closer together upto intermediate distances such as 14-16 Å with a broad distribution in the population (Table 1). The addition of excess EDTA decreased broadening in EPR spectra of doubly spin-labeled MalG511V16C/R129C (Figure 4, S5) and the Gaussian distribution for distance between two MalK domains could fit to a larger distance of >20Å with a narrow distribution relative to the nucleotidebound state or post-hydrolysis state (Table 1). Analogous experiments were also conducted with doubly spin-labeled MalG511-V16C/R129C in detergent micelles. Overall, the results were qualitatively similar to those in the proteoliposomal preparation (Figure S9A). Figure 4: EPR spectra of the doubly spin-labeled MalG511-V16C/R129C reconstituted in proteoliposomes with different ligands in the absence of MBP. Spectra were recorded in the absence (left) or presence (right) of maltose. Ligands were added sequentially. Spectra with different ligands are overlaid (top) and colored with designated colors; No ligand (black), + ATP + EDTA (red), + ATP + EDTA + MgCl2 (blue), + ATP + EDTA + MgCl2 + excess EDTA (green). Data is normalized to an equal number of spins (double integration of signal). Distance distributions, which were calculated by program ShortDistances106 [35, 36] for each condition, are shown with their designated colors (bottom). The x axis indicates distance in Å. Grey dotted line indicates the mean distance between MalK domains in specific condition. Individual spectra in the presence and absence of maltose are superimposed and shown in Figure S5. Absorption or first integral spectra in these conditions are shown in Figure S8A.


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Biochemistry

Figure 4

Table 1: Distance distributions of MalK domains of doubly spin-labeled MalG511-V16C/R129C The best fit to one Gaussian distribution was determined using the program ShortDistances106 [35, 36]. R is the peak distance (Å) and W is the width at half maximum of the Gaussian fit. % I and % NI correspond to percentage of interacting and non-interacting spins. Non-interacting spins include single spin-labeled proteins and doubly spin-labeled proteins in which spins are positioned far apart and out of the measurable range (10-25Å). Mean for each of these values are included. Distances below 10Å and above 20Å may not be accurate. χ2 Value defines the difference between observed and simulated spectra of doubly spin-labeled MalG511-V16C/R129C during their fitting. Sample MalG511-V16/R129C + no MBP

R W % %NI χ2 Value I

State In absence of maltose


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Resting state

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26 9

62 38

2.85E-09

Nucleotide Bound State

+ ATP + EDTA

18 20 68 32

1.97E-09

Post-hydrolysis State

+ ATP + EDTA + MgCl2

14 15 73 27

1.83E-09

Reversal Phase

+ ATP + EDTA + MgCl2 + excess EDTA

21 6

63 37

4.30E-09

Transition State

+ ATP + MgCl2 + (VO4)2-

8

1

70 30

5.49E-09

MalG511-V16/R129C + no MBP

In presence of maltose

Resting state

+ maltose

26 8

62 38

3.46E-09


+ maltose + ATP + EDTA

19 14 71 29

3.39E-09

Posthydrolysis State

+ maltose + ATP + EDTA + MgCl2

16 13 76 23

2.94E-09

Reversal Phase

+ maltose + ATP + EDTA + MgCl2 + excess EDTA

21 9

65 35

4.19E-09

Transition State

+ maltose + ATP + MgCl2 + (VO4)2-

9

1

72 28

5.45E-09

22 7

57 43

3.75E-09

MalG511-V16/R129C + MBP

In absence of maltose

Resting state Nucleotide Bound State

+ ATP + EDTA

15 17 66 34

3.78E-09



14 12 73 27

3.57E-09

Reversal Phase


17 13 71 29

5.41E-09

Transition State

+ ATP + MagCl2 + (VO4)2-

8

MalG511-V16/R129C + MBP

1

76 24

5.41E-09

53 47

5.25E-09


Resting state

+ maltose

19 5



9

23 70 30

3.14E-09



11 20 68 32

3.13E-09

Reversal Phase


14 14 73 27

4.86E-09

Transition State


8

5.50E-09


1

78 22

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Biochemistry

MalG511-V16/R129C + MBPG13D

In absence of maltose

Resting state

26 9

71 29

5.42E-09


+ ATP + EDTA

19 12 61 39

3.98E-09



15 11 71 29

3.68E-09

Reversal Phase


23 14 69 31

9.54E-09

Transition State

+ ATP + MagCl2 + (VO4)2-

8

MalG511-V16/R129C + MBPG13D

1

76 24

5.49E-09


Resting state

+ maltose

25 8

62 28

5.67E-09



18 14 65 35

4.30E-09



14 12 73 27

4.22E-09

Reversal Phase


21 6

54 28

1.62E-08

Transition State


8

73 27

5.42E-09

1

MBP stimulates the closure of MalK domains in MalG511 but inhibits its turnover To understand how high concentrations of MBP inhibit maltose accumulation by MalG511 as well as its ATPase activity, a similar set of EPR spectra of doubly spin-labeled MalG511V16C/R129C in proteoliposome preparations were obtained at higher concentrations of MBP (Figure 5, S6). Also, these EPR experiments at low concentrations of MBP were not feasible due to technical limitations. The concentration of doubly spin-labeled MalG511-V16C/R129C should be at least 60-80µM to obtain sufficient signal in the spectrum. And the concentrations of MBP should be more than double the amount of spin-labeled MalG511-V16C/R129C to obtain the complete response from MalG511-V16C/R129C and clearly observe changes in spectra.


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

Therefore, it was impractical to do this set of experiments at lower concentrations of MBP. This is why only higher concentrations of MBP were chosen for these EPR experiments. Figure 5: EPR spectra of the doubly spin-labeled MalG511-V16C/R129C reconstituted in proteoliposomes with different ligands in the presence of MBP. Spectra were recorded in the absence (left) or presence (right) of maltose. Ligands were added sequentially. Spectra with different ligands are overlaid (top) and colored with designated colors; No ligand (black), + MBP (yellow), + MBP + ATP + EDTA (red), + MBP +ATP + EDTA + MgCl2 (blue), + MBP +ATP + EDTA + MgCl2 + excess EDTA (green). Data is normalized to an equal number of spins (double integration of signal). Distance distributions, which were calculated by program ShortDistances106 [35, 36] for each condition, are shown with their designated colors (bottom). The x axis indicates distance in Å. Grey dotted line indicates the mean distance between MalK domains in the specific condition. Individual spectra in the presence and absence of maltose are superimposed and shown in Figure S6. Absorption or first integral spectra in these conditions are shown in Figure S8B. Figure 5

Addition of MBP alone in the absence of maltose caused slight broadening in EPR spectra of doubly spin-labeled MalG511-V16C/R129C (Figure 5, S6), in which MalK domains were far


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Biochemistry

(>20Å) apart with a distribution of 7 Å (Table 1). However, MalK domains came closer to intermediate distances in the presence of maltose with a narrow distribution in population (Figure 5, S6, Table 1). Addition of ATP and EDTA to spin-labeled MalG511-V16C/R129C in the presence of MBP resulted in a major broadening in its EPR spectra (Figure 5, S6), which corresponded to intermediate (15Å) and shorter distances (9Å) between MalK domains of the population in the absence or presence of maltose respectively (Table 1). The distribution was wide in the population for this nucleotide-bound state (Table 1). This result indicates that MBP facilitates a closure of the MalK domains upon the addition of ATP and EDTA. However, this closure is only partial, as MalG511 adopts a semi-open conformation with respect to MalK domains in the nucleotide-bound state which do not close to a distance of 7-8 Å, as observed in MalFGK2 [16, 20 Figure 2B, C] even in the presence of MBP (Figure 5, S6, Table1). When doubly spin-labeled MalG511-V16C/R129C in the presence of MBP was further stepped through its catalytic cycle by the addition of MgCl2 to reach the post-hydrolysis state, no changes were observed in its EPR spectra (Figure 5, S6). This shows that MalK domains in MalG511 remained close to intermediate distances (14Å or 11Å apart) with a wide distribution in the posthydrolysis state (Table 1). Even the addition of excess EDTA was unable to return the distance between MalK domains to larger distances (>20Å) and remained close to intermediate distances (17Å or 14Å) (Figure 5, Table 1). These results indicate that a high concentration of MBP prevents the turnover of ATP by MalG511 and a re-initiation of its catalytic cycle. In summary, these experiments indicate that MBP stimulates the partial closure of MalK domains in the MalG511 catalytic cycle but inhibits its turnover. Similar results were found with MBP and MalG511 in detergent preparation (Figure S9B). The same set of experiments was done in the presence of maltose and results were qualitatively similar.


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

MBPG13D suppressor variant restores turnover of MalG511 To investigate how MBPG13D affects the conformation of the MalG511 during its catalytic cycle, EPR spectra of doubly spin-labeled MalG511-V16C/R129C with different ligands were recorded in the presence of the MBP suppressor mutant, MBPG13D. The addition of MBPG13D to the doubly spin-labeled MalG511-V16C/R129C did not result in any pronounced effect on its EPR spectrum (Figure 6, S7). MalK domains in the majority of MalG511 population were very far (>20Å) apart from each other (Table 1). The addition of ATP and EDTA caused a major broadening in the EPR spectrum. This broadening in EPR sprectrum further increased when Mg2+ was supplied to advance the transporter to the post-hydrolysis state (Figure 6, S7). Distance measurements showed that the MalK domains were within intermediate (18Å) and shorter (14Å) distances of one another, in the nucleotide-bound state and post-hydrolysis states, respectively, both in the presence and absence of maltose (Table 1). Again, the distance distribution was wide. However, with the addition of excess EDTA, MBPG13D, unlike MBP with MalG511, increased the distance between MalK subunits to larger ones (>20Å) (Table 1, Figure 6, S7). The results obtained with proteoliposome and detergent preparation were similar (Figure S9C), irrespective of the presence of maltose. Figure 6: EPR spectra of the doubly spin-labeled MalG511-V16C/R129C reconstituted in proteoliposomes with different ligands in the presence of MBP. Spectra were recorded in the absence (left) or presence (right) of maltose. Ligands were added sequentially. Spectra with different ligands are overlaid (top) and colored with designated colors; No ligand (black), + MBPG13D (orange), + MBPG13D + ATP + EDTA (red), + MBPG13D +ATP + EDTA + MgCl2 (blue), + MBPG13D +ATP + EDTA + MgCl2 + excess EDTA (green). Data is normalized to an equal number of spins (double integration of signal). Distance distributions, which were calculated by program ShortDistances106 [35, 36] for each condition, are shown with their designated colors (bottom). The x axis indicates distance in Å. Grey dotted line indicates the mean distance between MalK domains in the specific condition. Individual spectra in the presence and absence of


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Biochemistry

maltose are superimposed and shown in Figure S7. Absorption or first integral spectra in these conditions are shown in Figure S8C. Figure 6

MalG511 utilizes an alternating access mechanism in its catalytic cycle EPR experiments with doubly spin-labeled MalG511-V16C/R129C in the absence or presence of MBP or MBPG13D showed that the MalK domains of the MalG511, unlike those of the MalFGK2, did not approach within 7-8Å during the nucleotide-bound step of the transporter reaction cycle [16, 20 Figure 2B, C]. This finding raised the question of whether MalG511 utilizes the same alternating access model as MalFGK2 as a part of its transport cycle in the presence of MBP. Attempts were made to capture the closed transition state by vanadate trapping [37] of MalG511 in the absence or presence of MBP or MBPG13D. A major broadening in the EPR spectrum of doubly spin-labeled MalG511-V16C/R129C was observed upon vanadate trapping


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

in all three cases (Figure 7, S10). Calculated distances in the vanadate-trapped state showed that the MalK domains were very close (8Å) to each other with a very narrow distribution in population. This result implies that MalG511 also transitions between inward- and outward-open states as a part of alternating access mechanism in its catalytic cycle. Figure 7: Vanadate trapping of doubly spin-labeled MalG511-V16C/R129C in the absence (red spectrum) or presence of MBP (blue spectrum) or MBPG13D (green spectrum). The EPR spectrum of doubly spin-labeled MalG511-V16C/R129C without any ligand (black) is also shown. Spectra were recorded in the absence (left) or presence (right) of maltose. Data is normalized to an equal number of spins (double integration of signal). Distance distributions, which were calculated by program ShortDistances106 [35, 36] for each condition, are shown with their designated colors (bottom). The x axis indicates distance in Å. Grey dotted line indicates the mean distance between MalK domains in the specific condition. Individual spectra in the presence and absence of maltose are superimposed and shown in Figure S10. Figure 7


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Biochemistry

N-lobe of MBP remains anchored to MalG511 but C-lobe plays dynamics with it throughout its catalytic cycle. EPR spectroscopy was further used to understand specific MBP-transporter interactions to get an in-depth understanding of inhibition of MalG511 by MBP. Residues D41 and S352 in the N-lobe and C-lobe of MBP (Figure 8A and 8B) respectively, were separately mutated to cysteine and spin labeled [22, 23]. Both of these MBP mutants were stepped through the transporter catalytic cycle, first by the addition of ATP in the presence of EDTA and then by the addition of MgCl2. Figure 8: [A and B] Location of cysteine mutations (D41 in N-lobe and S352 in C-lobe) on MBP for site directed spin labeling in EPR spectroscopy. MBP is in magenta and open conformation (PDB: 3FH6, Chain E [16]) (A) viewed from the interface that interacts with MalFGK2 (B) side view as it interacts on the top of MalFGK2. Residues D41 and S352 are colored in cyan and orange respectively. [C and D] EPR spectra of spin-labeled (C) MBPD41C and (D) MBPS352 with the MalG511 reconstituted in nanodiscs (MalG511-n) and different ligands. Spectra were recorded in the absence (left) or presence (right) of maltose. Ligands were added sequentially. Spectra with different ligands are overlaid and colored with designated colors; No ligand (black), + MalG511-n (red), + MalG511-n + ATP + EDTA (blue), + MalG511-n +ATP + EDTA + MgCl2 (green), + MalG511-n +ATP + EDTA + MgCl2 + excess EDTA (yellow). Data is normalized to an equal number of spins (double integration of signal). Individual spectra in the presence and absence of maltose are superimposed and shown in Figure S11. Figure 8


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

EPR spectra of spin-labeled MBPD41C in the absence of ligands had a line shape characteristic of a relatively mobile spin label on the surface of a protein (Figure 8C) [38]. The lineshape of spin-labeled MBPS352 was consistent with that of a free spin label on a helical surface (Figure 8D) [39]. A minimal decrease in mobility was observed upon the addition of MalG511 to the MBP spin-labeled at D41 in proteoliposomal preparations. The spectra remained unchanged during all steps in the catalytic cycle (data not shown). Similar results were obtained with MBP spin-labeled at S352 and MalG511 (data not shown). These results might be explained by limited


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Biochemistry

accessibility of ligands to both sides of the membrane in the vesicles. To address this issue, we reconstituted MalG511 in nanodiscs [33]. Nanodiscs are a novel class of membrane mimicking media that consist of a segment of phospholipid bilayer surrounded by membrane scaffold protein (MSP) of defined and controllable size [40]. A membrane protein can be incorporated into this patch of membrane and can be accessible to aqueous solution through both of its hydrophilic surfaces [41]. Nanodiscs have been proven to be a convenient alternative to liposomes to assess protein-protein interaction and conformational changes in the maltose transporter by EPR in a membrane environment [33]. EPR spectra of spin label attached to D41 showed a major broadening accompanied by an increase of the immobile component upon addition of MalG511 in nanodiscs preparations (Figure 8C, S11A). The spectrum remained the same upon the addition of ATP and EDTA as well as after the addition of MgCl2. The presence of maltose had no effect on the spectrum. Also, the addition of excess EDTA to revert to the resting state did not alter the spectrum. Similar EPR experiments were carried out with MBP spin-labeled at position 352 (Figure 8D, S11B). It also showed a significant increase in the immobile component of its spectra upon the addition of the MalG511. The magnitude of this immobile component was further intensified with the addition of maltose transporter ligands and became very prominent in the post-hydrolysis state. No further changes occurred if excess EDTA was provided (Figure 8D, S11B). The presence of maltose further increased the immobile component of spin-labeled MBPS352C (Figure 8D, S11B black spectra). The results of EPR experiments with both spin-labeled MBPD41C and MBP352C using MalG511 in detergent phase (Figure S12) were very similar to results obtained in nanodiscs.


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Page 28 of 41

DISCUSSION Site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy have been proven to be robust methods to study the conformational dynamics (open, semi-open and closed states) of MalFGK2 and its interaction with MBP at different stages throughout the catalytic cycle [20-23, 26, 42]. We also used the same tool to study the catalytic cycle of MalG511 in the absence or presence of MBP or MBPG13D. Spin labels were attached to residues in MalK domains to understand the opening and closing of the MalK interface during the catalytic cycle of MalG511. Calculated distances between spin labels attached to MalK domains have been summarized in Table 2. These distances show that MalG511 transitions into three different states (open (>20Å), semi-open (10-18Å) and closed (8Å) states) during its catalytic cycle. Table 2: Summary of distances (in Å) between the spin labels attached at V16 and R129 positions in MalK domains during different stages in the catalytic cycle of MalG511 reconstituted in proteoliposomes (from Table 1). Steps in Catalytic cycle

Ligands

MalFGK2 + MBP

MalG511

MalG511 + MBP

MalG511 + MBPG13 D

Resting State

-

>20

>20

>20

>20


ATP + EDTA

7-8

b

18/19

Post-hydrolysis State

ATP + EDTA + MgCl2

10-14

b

14/15

Reversal Phase

ATP + EDTA + MgCl2 + excess EDTA

>20

>20

Vanadate Trapping

ATP + MgCl2 + (VO4)2-

7-8

8.4

a


b

14/9

18

b

14/10

14

b

17/14

>20

8.2

8.3

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Biochemistry

a Distances between MalK domains of MalFGK2 in the presence of MBP are taken from [20]. b Numbers indicate distances in absence/presence of maltose. In the resting state, when no ligand is bound to MalG511, it exists in an inward facing open state in which the MalK domains are far apart (>20Å) from each other, irrespective of the presence of MBP. When MBP is not present, MalG511 transitions into a semi-open conformation (18Å) in nucleotide-bound state. This state is very similar to the pretranslocation state of MalFGK2 which is observed when the transporter is in complex with both AMP-PNP and a locked-closed MBP [21]. During ATP hydrolysis, MalK domains of MalG511 come close together to intermediate distances (14Å), which is again a semi-open state, similar to the post-hydrolysis state of MalFGK2 [20 Figure 2B, C]. A broad distribution of MalG511 in nucleotide-bound and posthydrolysis states (Table 1) suggests that MalG511can exist in multiple conformations in the semi-open state, when MalK domains are very flexible. Since these results were obtained in the absence of MBP, these findings suggest that MalG511 can undergo a series of conformational changes during its catalytic cycle, even in the absence of MBP. This observation explains the high basal activity of MalG511 in the absence of maltose and MBP. In the presence of MBP, MalG511 also transitions to a semi-open nucleotide-bound and post-hydrolysis state (14-15Å). In the presence of maltose, the distribution of the semi-open population is shifted to shorter distances (9-10Å) in MalK domains. Both of these results suggest that MBP and maltose promote the closure of the MalK domains in MalG511. MalG511 could not revert back to its resting open state during the reversal phase, which indicates that MBP, at high concentrations, prevents the turnover of the MalG511 transporter and re-initiation of its catalytic cycle. Collectively, these experiments indicate that MBP stimulates the partial closure of MalK domains in the MalG511


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

catalytic cycle but also inhibits its turnover at higher concentrations. It explains the unusual biphasic behavior of MalG11 in maltose transport assays [29] as well as ATPase assays in the presence of MBP. Similar open and semi-open states were found in the catalytic cycle of MalG511 with MBPG13D as observed in the absence of MBP. However, in this case, during the reversal phase of the catalytic cycle, MalG511 could convert back to an open state, which was not observed in the presence of MBP. This behavior explains the restoration of the hyperbolic behavior in maltose transport assays [29] as well as ATPase assays with MalG511 in the presence of MBPG13D. Besides open and semi-open states, MalG511 also adopts a closed transition state with vanadate trapping in the absence or presence of MBP or MBPG13D. In this state, MalK domains are very close (8Å) to each other. These results support that MalG511 uses an alternating access mechanism like other ABC transporters. Spin labels on residues D41 and S352 on the N- and C-lobes of MBP, respectively, were used to study interactions between MalG511 and MBP using EPR spectroscopy. Broadening of the spectra of a spin label attached to either D41 and S352 with addition of MalG511, in absence of any ligands, suggests a strong interaction between MBP and MalG511. Slight changes in the spectra of MBP spin-labeled at D41 on the N-lobe with the addition of the series of ligands suggest that the N-lobe of MBP remains tightly bound to the MalG511 throughout the transporter catalytic cycle. However, the interaction of the C-lobe of MBP with MalG511 changes during its catalytic cycle and is also affected by the presence of maltose, suggesting that C-lobe interactions determine the substrate specificity of MalG511 [22]. The immobile component of the spin label attached to MBPS352 was more prominent with detergent preparations, which may imply different regulation of MalG511 by its substrate in membrane media. While the N-lobe of MBP remains anchored to MalG511, the C-lobe interacts dynamically with the transporter throughout its cata-


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Biochemistry

lytic cycle. In addition, the spin label attached to both lobes remains immobile during the reversal phase, which is consistent with unproductive interactions between MalG511 and MBP in this step and an inhibitory role of MBP in the catalytic cycle of MalG511. A plausible model for the catalytic pathway of the MalG511 in the absence or presence of MBP would include many of the same steps used by the MalFGK2 (Figure 9) [22]. In the absence of ATP and MBP, MalG511 exists in an open resting state. The addition of ATP either induces a conformational change to promote an active state [43] or stabilizes an active conformation that pre-exists in equilibrium with the resting state (Figure 9, Step A). Although the addition of MBP is not required for the active state to dominate, when MBP is present the active state is further stabilized in a pretranslocation semi-open state (Figure 9, Step B). MalK domains then come close together in an unstable transition state to harness the energy from ATP hydrolysis (Figure 9, Step C), which converts into a stable semi-open posthydrolysis state (Figure 9, Step D). This semi-open state reverts back to resting open state with the release of ADP (Figure 9, Step E). However, when MBP is present, MalG511 interacts unproductively with MBP and cannot revert back. In contrast, the MBP suppressor mutant - MBPG13D restores the functioning of MalG511 by supporting its turnover. Figure 9: Model depicting the catalytic cycle of MalG511. Color coding: MalF, blue rectangle; MalG, pink rectangle; MalK domains, brown ovals; Spin labels on V16 and R129 on MalK domains are labeled as small circles with cyan and orange color, respectively; ATP are grey ovals; MBP, yellow shape; Spin labels on D41 in N-lobe and S352 in C-lobe of MBP are labeled as small circles with green and purple color, respectively; Red circle, maltose. MalG511 undergoes a cycle of events to couple maltose transport with ATP hydrolysis. MalK domains start closing with binding of ATP in the nucleotide-bound state [A], and close further upon binding of MBP [B]. At this point, MalG511 passes through a closed transition state which can be trapped by vanadate [C]. This is followed by ATP hydrolysis [D] and a return back to the resting state [E]. This final reversal step occurs only in the absence of MBP or presence of MBPG13D, as MalG511 does not readily release MBP to complete the catalytic cycle.


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

Figure 9

EPR data with doubly spin-labeled MalG511-V16C/R129C and spin-labeled MBP suggest that the origin of the biphasic nature of the MalG511 ATPase activity may involve the posthydrolytic steps. A kinetic model proposed by Kuhl termed the "Recovery model" might explain the unusual activity profile of MalG511 [44]. According to this model, MalG511 undergoes an obligatory recovery period following the ATP hydrolytic step. Presumably, MalFGK2 rapidly resets to the resting state and can re-enter the catalytic pathway. However, if MalG511 returns to the resting state more slowly, MBP at higher concentrations would have more chances to bind, leading to substrate inhibition and decreased activity due to unproductive consumption of active MalG511.


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Biochemistry

Comparing the catalytic cycle of MalFGK2 and MalG511, these results show that MalG511 uses many of the same steps in its catalytic reaction cycle as MalFGK2, but does so in the absence of liganded MBP [22]. Here, a key difference between the catalytic cycle of MalFGK2 and MalG511 is that MalG511 does not require MBP to go into a closed transition state. In contrast, MalFGK2 essentially requires MBP to proceed towards a closed transition state (7-8Å) [20 Figure 2B, C]. MalFGK2 does not show any change in distance between the MalK domains when bound to ATP in the absence of MBP. The MalK dimer in MalFGK2 closes to within 7-8Å (closed transition state) but only in the presence of both MBP and ATP [20 Figure 2B, C]. However, the semi-open states in the pre-hydrolysis and post-hydrolysis stages in MalFGK2, in the presence of MBP bound with maltose and MBP alone [21, 22], respectively, are similar to semiopen states in the catalytic cycle of MalG511, irrespective of the presence of maltose or MBP. The equilibrium of MalG511 is shifted more towards a semi-open state, which makes it difficult to observe the closed transition state, unless it is trapped by a transition state analog such as vanadate. However, MalFGK2 can easily adopt the closed state in the presence of ATP and MBP bound with maltose. This shift in equilibrium towards a semi-open state is very much consistent with our hypothesis [10]. It has been hypothesized that the ground open state and semi-open pretranslocation (active state) conformations in the catalytic cycle of MalFGK2 exist in equilibrium [22]. MalFGK2 predominantly occupies the ground state in the absence of MBP, but then switches to the active transition state when MBP and ATP are present. We demonstrate that the mutations in MalG511 are believed to stabilize the mutant transporter in a semi-open conformation that more closely resembles the transition state during the catalytic cycle. As a result, the energy barrier of the enzymatic reaction is lowered by affecting the equilibrium constant between the two conformational states [10, 29]. The data reported in this study is consistent with the idea that


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

this shift in equilibrium allows MalG511 to bind and hydrolyze ATP in the absence of MBP. As a result, MalG511 would not require the binding energy of MBP to assume the semi-open pretranslocation state that necessitates liganded MBP in MalFGK2 [21, 22]. We also show that binding of MBP and ATP is insufficient to fully drive the MalG511 to the closed transition state, as in the case of MalFGK2. The inability to achieve the closed state may reflect a less productive interaction of MBP with MalG511. Nevertheless, the transition state analog vanadate binds sufficiently tightly to force the closure of the MalK domains. In summary, the data presented here are consistent with MalG511 utilizing a common alternating access mechanism employed by other members of the ABC transporter superfamily. Elucidating the molecular mechanism of the binding-protein-independent mutant, MalG511 is significant as it may mirror the mechanism of altered disease-related mutants in the ABC transporter superfamily. For example, ABC exporters like P-glycoprotein and cystic fibrosis transmembrane conductance regulator, which are implicated in multidrug resistance and cystic fibrosis, respectively, exhibit high basal activity and also do not utilize any binding protein to transport their substrate [31]. P-glycoprotein is an ATP powered drug efflux pump linked to multidrug resistance in mammalian cell lines and human cancers. P-glycoprotein interacts with a variety of nonpolar substrates and displays high levels of constitutive ATPase activity (3-5 mol/min/mg protein), even in the absence of its substrate [45]. Moreover, the basal activity of Pglycoprotein, like that of MalG511, exhibits a biphasic pattern in response to some of its drug substrates, with ATPase activity being stimulated at low concentrations and inhibited at higher concentrations [46, 47]. These complex patterns of modulation of P-glycoprotein ATPase activity, which are also dependent on detergent and lipids, may follow the similar relationship as we observed in this study between MalG511 and MBP.


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Biochemistry

To conclude, this study has elucidated the catalytic cycle of MalG511, a binding-proteinindependent mutant of maltose transporter, in the absence or presence of MBP or its suppressor mutant, MBPG13D, using SDSL-EPR spectroscopy. These results can help us to understand mechanistic regulation in other disease-related mutants in ABC transporter family and so facilitate future drug design. Supporting Information Basal ATPase activity of MalG511 in detergent (Figure S1), Distance measurement using shortdistances106 (Figure S2), EPR spectra of singly spin-labeled MalG511-V16C (Figure S3), MalG511-R129C (Figure S4) and doubly spin-labeled MalG511-V16C/R129C (Figure S5, S6, S7) reconstituted in proteoliposomes with different ligands in the absence or presence of MBP or MBPG13D, Absorption spectra of doubly spin-labeled MalG511-V16C/R129C reconstituted in proteoliposomes (Figure S8), EPR spectra of the doubly spin-labeled MalG511-V16C/R129C in detergent (Figure S9), Vanadate trapping of doubly spin-labeled MalG511-V16C/R129C (Figure S10), EPR spectra of spin-labeled MBPD41C and MBPS352 with the MalG511 reconstituted in nanodiscs (Figure S11) or detergent (Figure S12).


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AUTHOR INFORMATION Corresponding Author Ruchika Bajaj, Hockmeyer Hall of Structural Biology, Department of Biological Sciences, Purdue University. Email address: [email protected] Present Address Departments of Bioengineering & Therapeutic Sciences and Pharmaceutical Chemistry, Rock Hall, Mission Bay Campus, University of California San Francisco Author Contributions Ruchika Bajaj and Amy L. Davidson designed experiments and analyzed results. Mariana I Park produced preliminary results. Ruchika Bajaj planned and executed all experiments, interpreted results and wrote the manuscript, which was improved further with contributions from all authors. ACKNOWLEDGEMENTS We would like to thank Dr. Christian Altenbach for his guidance in the ShortDistances106 program. We also thank Prof. Frederick S. Gimble, Prof. Robert M. Stroud, Prof. Deanna L. Kroetz, Dr. Cedric Orelle and Dr. Frances Joan D. Alvarez for their valuable comments and help during preparation of the manuscript. This work is dedicated to the memory of my advisor Prof. Amy L. Davidson. Funding Information The study was supported by NIH Grant GM070515.


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Conformational dynamics in binding-protein-independent mutant of

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