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Structural basis for the Serratia marcescens lipase secretion system: Crystal structures of the membrane fusion protein and nucleotide-binding domain Daichi Murata, Hiroyuki Okano, Clement Angkawidjaja, Masato Akutsu, Shun-ichi Tanaka, Kenyu Kitahara, Takuya Yoshizawa, Hiroyoshi Matsumura, Yuji Kado, Eiichi Mizohata, Tsuyoshi Inoue, Satoshi Sano, Yuichi Koga, Shigenori Kanaya, and Kazufumi Takano Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00985 • Publication Date (Web): 02 Nov 2017 Downloaded from http://pubs.acs.org on November 7, 2017

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Biochemistry

Structural basis for the Serratia marcescens lipase secretion system: Crystal structures of the membrane fusion protein and nucleotide-binding domain

Daichi Murata†, Hiroyuki Okano‡, Clement Angkawidjaja‡, Masato Akutsuǁ, Shun-ichi Tanaka∫, Kenyu Kitahara†, Takuya Yoshizawa∫, Hiroyoshi Matsumura∫, Yuji Kado‡, Eiichi Mizohata‡, Tsuyoshi Inoue‡, Satoshi Sano†, Yuichi Koga‡, Shigenori Kanaya‡, Kazufumi Takano*†

† Department of Biomolecular Chemistry, Kyoto Prefectural University, Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522 Japan ‡ Graduate School of Engineering, Osaka University, Yamadaoka, Suita 565-0871, Japan ǁ Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Max-von-Laue-Straße, 60438 Frankfurt am Main, Germany ∫ College of Life Sciences, Ritsumeikan University, Noji-Higashi, Kusatsu 525-8577, Japan

* Corresponding author E-mail address of the corresponding author: [email protected] Tel/Fax of the corresponding author: +81-75-703-5654

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ABSTRACT: Serratia marcescens secretes a lipase, LipA, through a type I secretion system (T1SS). The T1SS for LipA, the Lip system, is composed of an inner membrane ABC transporter with its nucleotide-binding domains (NBD), LipB, a membrane fusion protein, LipC, and an outer membrane channel protein, LipD. Passenger protein secreted by this system has been functionally and structurally characterized well, but relatively little information is available on the transporter complex. Here, we report the crystallographic studies of LipC without the membrane anchor region, LipC-, and the NBD of LipB (LipB-NBD). LipC- crystallographic analysis has led to the structure determination of the long α-helical and lipoyl domains, but not the area where it interacts with LipB, suggesting that the region is flexible without LipB. The long α-helical domain has three α-helices, which interacts with LipD in the periplasm. LipB-NBD has the common overall architecture and ATP hydrolysis activity of ABC transporter NBDs. Using the predicted models of full-length LipB and LipD, the overall structural insight of Lip system is discussed.


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INTRODUCTION In Gram-negative bacteria, proteins translocated to the extracellular medium have to pass two permeability barriers, the inner and outer membranes, with the periplasm in-between. Several systems have been identified,1-5 one of which is the type I secretion system (T1SS) that belongs to the tripartite efflux assemblies, which include the multidrug efflux (MDR) pumps related to the export of various molecules.6-9 Secretion by this system is done in a single, energy-coupled step, resulting in direct passage across both inner and outer cell membranes and the intervening periplasm. This system is composed of three protein subunits, an inner membrane protein (IMP, an ABC transporter), a membrane fusion protein (MFP, or periplasmic adaptor proteins (PAP)), and an outer membrane channel protein (OMP, or outer membrane factor (OMF)). ABC transporter is located at the inner membrane and is responsible for the generation of energy needed for transport, resulting from ATP hydrolysis by its nucleotide-binding domains (NBD). The initial recognition of passenger proteins also takes place in this subunit. MFP forms a complex with the ABC transporter in the periplasm and plays a role in the folding of the passenger protein, as shown in Escherichia coli hemolysin (HlyA) secretion system that consists of HlyB as ABC transporter, HlyD as MFP and TolC as OMP.10,11 OMP is best represented by TolC, which forms a homotrimer and a channel with a long axis measuring approximately 140 Å and an internal diameter of 35 Å, protruding the periplasm and providing the ‘tubing’ for the transport.12 TolC is responsible for the multidrug transport and HlyA secretion in E. coli.10,13 The passenger proteins of T1SS range from RTX (repeat in toxin) such as HlyA, Bordetella pertussis adenylate cyclase14 and Pasteurella haemolytica leukotoxin,15 to extracellular enzymes such as Erwinia chrysanthemi protease16 and Serratia marcescens 3 ACS Paragon Plus Environment

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lipase (LipA),17 as well as proteins that remain attached to the cell surface such as S-layer proteins18 and certain glycanases.13 The passenger proteins usually have a secretion signal located within the last 60 residues from the C-terminus, which is not cleaved after secretion.19 Repetitive sequences, termed the RTX motif, composed of nine amino acids, GGxGxDxux (x: any amino acid, u: hydrophobic amino acid), are also present at the upstream region of the secretion signal in some passenger proteins such as HlyA and LipA.20 These repeats bind Ca2+ ions and form a β-roll motif, as shown by the crystal structures of passenger proteins.21-23 LipA (Uniprot code: Q59933) is a family I.3 lipase, and its secretion machinery consists of LipB (Uniprot code: Q54456) as an ABC transporter, LipC (Uniprot code: Q54457) as a MFP and LipD (Uniprot code: Q54458) as an OMP.20 LipA has two domains: an N-terminal catalytic domain and a C-terminal domain with the RTX motif and a secretion signal. Pseudomonas sp. MIS38 lipase (PML, Uniprot code: Q9RBY1) is a homolog of LipA with 61% amino acid sequence similarity.24,25 Co-expression of these lipases with their cognate T1SS in E. coli has been shown to permit the secretion of the lipases.26,27 Furthermore, these lipases could also be secreted by a heterologous T1SS. PML is efficiently secreted via LipA’s cognate T1SS, the Lip system or LipB-LipC-LipD, in E. coli. The hybrid system has so far been used for the systematical secretion analysis of PML and its derivatives.28,29 The C-domain of PML could be used as a secretion tag for extracellular production of a heterologous protein via the Lip system.30 Mutational, crystallographic, and MD simulation studies on PML revealed its interfacial activation mechanism.31-35 In contrast to the well-characterized passenger proteins, structural features of Lip system remain to be elucidated.


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Although the crystal structures of some constituent proteins in T1SS, such as TolC,12 HlyB-NBD,36 HlyD soluble region,37 PrtD from Aquifex aeolicus,38 have been already determined, T1SS structural information is considerably limited. Here, to further elucidate the structural basis of T1SS, we determined the crystal structures of the long α-helical and lipoyl domains of LipC (Asn62-Ser327) and the NBD of LipB (LipB-NBD, Lys301-Asn588). The results contribute to the structural insights of the Lip system.

MATERIALS AND METHODS Expression and Purification. pYBCD20 containing lipBCD was constructed previously.18 Membrane anchor site of LipC (aa 26-46) was predicted by using a hydropathy plot (http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form=topped), and pYBCD20 was used as a template for constructing derivative plasmids containing lipC-, a LipC derivative with its membrane anchor deleted (Met1-Arg25 and Leu47-Glu443) and lipB-NBD by using Quick Change II XL Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA). The genes of lipC- and lipB-NBD were amplified using the derivative plasmids as a template by PCR, and then this amplified genes were inserted into the NdeI and XhoI sites of plasmid pET-25b(+) and pET-28a (Novagen,

Darmstadt,

pET-28a/LipB-NBD

Germany),

was

used

respectively, as

a

for

template

overproduction. for

constructing

Plasmid plasmid

pET-H525A-LipB-NBD used to overproduce H525A variant of LipB-NBD. BL21(DE3)-CodonPlus cells, transformed with pET25b(+)-LipC-, were grown in NZCYM medium containing 50 mg/L ampicillin at 37°C up to OD600=0.5, and protein 5 ACS Paragon Plus Environment

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production was induced by the addition of IPTG (final concentration of 1 mM) and further culturing for 5 h at 37°C. The culture was centrifuged at 10,000×g for 20 min at 4°C, and the precipitant was resuspended in a lysis buffer (20 mM phosphate buffer pH 7.4, 300 mM NaCl, and 5% glycerol). After sonication and centrifugation at 30,000×g for 30 min at 4°C, the supernatant was preliminarily purified by 40% ammonium sulfate precipitation. Following centrifugation at 10,000×g for 20 min at 4°C, the precipitate was subsequently resuspended with the same lysis buffer, followed by dialysis against the same buffer supplemented 10 mM imidazole (buffer A). The mixture was gently mixed with Ni-NTA resin (GE Healthcare, Chicago, IL) equilibrated with buffer A. The resin was washed using 40 mM imidazole in lysis buffer, and the protein was eluted with 300 mM imidazole, followed by dialysis against 5 mM phosphate buffer pH 6.8. The protein was subjected to hydroxyapatite column (GE Healthcare) equilibrated with 5 mM phosphate buffer pH 6.8. The column was washed using the same phosphate buffer, and the protein was eluted with a linear gradient of 5-300 mM phosphate buffer pH 6.8. The fractions containing the protein was then subjected to gel-filtration chromatography using 16/60 Superdex 200 column (GE Healthcare) equilibrated with 10 mM Tris-HCl pH 7.5 with 150 mM NaCl. The protein was eluted as a dimer. The purified protein was dialyzed against 10 mM Tris-HCl pH 7.5 and concentrated to 9.0 mg/ml using Amicon centrifugal filter device (Millipore, Billerica, MA) with 10 kDa MW cut-off. BL21(DE3) cells, transformed with pET-28a/LipB-NBD or pET-H525A-LipB-NBD, were grown in NZCYM medium containing 50 mg/L ampicillin at 37°C up to OD600=0.5, and protein production was induced by the addition of IPTG (final concentration of 1 mM) and shaking culture for 4 h at 25°C. The culture was 6 ACS Paragon Plus Environment

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centrifuged at 6,000×g for 10 min at 4°C, and then the precipitant was resuspended with a lysis buffer (25 mM phosphate buffer pH 8.0, 100 mM KCl, 20% glycerol, 1 mM phenylmethylsulfonyl fluoride and 10 mM imidazole). Following French press and centrifugation at 160,000×g for 1 hour at 4°C, the supernatant was applied to a HiTrap Chelating column (5 ml) (GE Healthcare) equilibrated with the same buffer. The protein was eluted from the column with a linear gradient of imidazole from 10 to 300 mM. The fractions containing the protein were collected, dialyzed against 10 mM CAPS-NaOH pH 10.4 containing 20% glycerol. The sample was concentrated and applied to a 16/60 Superdex 200 pg column (GE Healthcare) equilibrated with the dialysis buffer. The protein was eluted as a monomer. The fractions containing the protein were collected and used for further studies. The purity of the proteins was analyzed by SDS-PAGE on a 12% (w/v) polyacrylamide gel, followed by staining with Coomassie Brilliant Blue. The purified proteins were shown as single bands on SDS-PAGE. Protein concentrations were determined from the UV absorption on the basis that the absorbance at 280 nm of a 0.1% solution is 0.35 and 0.59 for LipC- and Lip-NBD, respectively. These values were calculated by using ε of 1576 M-1cm-1 for Tyr and 5225 M-1cm-1 for Trp at 280 nm.39 Crystallization and Structure Determination. Purified LipC- and LipB-NBD were concentrated to 9 mg/ml in 10 mM Tris-HCl pH 7.5 and 10 mg/ml in 10 mM CAPS-NaOH pH 10.4 containing 20% glycerol, respectively, using Amicon centrifugal filter device. The crystallization conditions were initially screened using crystallization kits from Hampton Research (Alise Viejo, CA; Crystal Screen I and II) and Emerald Biostructures (Bainbridge Island, WA; Wizard I and II) by the sitting-drop vapor-diffusion method at 4 and 20°C. Drops were prepared by mixing 1 µl each of 7 ACS Paragon Plus Environment

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protein and reservoir solutions and were equilibrated against a 100 µl reservoir solution. The largest LipC- crystals (0.4 mm × 0.1 mm × 0.1 mm) were obtained in one week using 32% ethylene glycol at 20°C. Cuboid-shaped crystals of LipB-NBD appeared after one week in Wizard I No.20 [1.6 M K2HPO4, 0.4 M NaH2PO4, 0.1 M imidazole pH 8.0, and 0.2 M NaCl] at 4°C. To improve the crystal quality, the crystallization conditions were further optimized. Diffraction-quality crystals grew after two weeks, when the protein concentration was increased to 15 mg/ml and the drop was prepared by mixing 2 µl protein solution with 2 µl reservoir solution [1.4 M K2HPO4, 0.6 M NaH2PO4, 0.1 M imidazole pH 8.0, and 0.2 M NaCl] at 4°C. LipC- crystals were cryoprotected by the addition of 35% ethylene glycol and were flash-cooled in liquid nitrogen. Diffraction data of native LipC- were collected at wavelength of 1.0 Å at -100°C (173 K) at beam line BL44XU in SPring-8 (Hyogo, Japan) and were processed with HKL2000.40 Moreover, derivative crystals were obtained by soaking the native crystals with the mother liquor containing 5 mM K2PtCl6 for 10 min. Diffraction data from the crystal of Pt derivative were collected at wavelength of 1.072 Å at -100°C at the beam line BL26XU in SPring-8 and were processed with XDS.41 The structure was determined by using autoSHARP in CCP4 suite42 using the processed data of native and Pt(IV) derivative. To find the Pt(IV) sites, autoSHARP tested SIRAS (single isomorpous replacement with anomalous signal) and SAD (single-wavelength anomalous diffraction) methods using high resolution limits of 6 Å with SHELXC/SHELXD and generated two initial heavy atom positions by SAD. SHARP program further refined the heavy atom positions. Density modification using 56.3% solvent content increased the overall figure of merit from 0.1 to 0.72. The initial model was built automatically by AutoBuild in Phenix.43 Further manual model 8 ACS Paragon Plus Environment

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building and refinement were performed with COOT,44 phenix.refine45 and REFMAC5 of

the

CCP4

suite.

Non-crystallographic

symmetry

(NCS)

and

TLS

(Translation/Libration/Screw) during refinement of LipC structure were used, however the electron density of one of the lipoyl domain could not be improved very much. The weak electron density of this moiety indicates a high mobility of the lipoyl domain, maybe because of the flexible future of the linker region between the lipoyl domain and the α-helical domain of LipC. We did not see obvious electron density of water molecules during structure refinement and did not add water in the model. Data collection and refinement statistics are shown in Table 1. X-ray diffraction data set of LipB-NBD was collected at a wavelength of 0.9 Å at -100°C at beam line BL44XU in SPring-8. The data set was indexed, integrated and scaled using the program HKL2000.40 The structure was determined by molecular replacement method using MOLREP in the CCP4 program suite.42 The 2.6 Å structure of HlyB-NBD (PDB: 1MT0) was used as a starting model as it is. Automated model building was done by using ArpWarp.46 Structural refinement was performed using REFMAC of the CCP4 suite42 and the model was corrected using COOT.44 NCS and TLS refinement methods were not used throughout the process. Water molecules were added using COOT. Data collection and refinement statistics are shown in Table 1. The figures were prepared using PyMol (http://www.pymol.org).


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Table 1. Data collection and refinement statistics of LipC- and LipB-NBD. Crystal

LipC-

LipC-_Pt

LipB-NBD

Wavelength (Å)

1.000

1.070

0.900

P32

P32

P21

a, b, c (Å)

126.31, 126.31, 71.41

124.14, 124.14, 72.15

72.30, 61.30, 88.31

α, β, γ (°)

α=γ=90.00, β=120.00

α=γ=90.00, β=120.00

α=γ=90.00, β=104.03

Space group Cell parameters

Molecules/asymmetric

2

2

unit Resolution range (Å)

50-2.90 (2.95-2.90)a

47.05-3.35 (3.53-3.35)

50-2.65 (2.70-2.65)

Reflections measured

185,557

193,692

307,001

Unique reflections

28,216

17,934

22,042

6.6 (6.6)

5.4 (5.6)

4.6 (3.8)

Completeness (%)

99.9 (100)

100 (100)

98.7 (90.5)

Rmerge (%)b

7.1 (89.4)

9.1 (79.3)

8.1 (49.7)

Average I/σ (I)

28.1 (2.0)

15.6 (3.1)

18.1 (1.5)

Redundancy

Wilson B-factor (Å2)

82.5

63.7

Phasing statistics FOM

0.1

FOM after DM

0.72

Refinement statistics Resolution limits (Å)

50-2.90

40-2.65

Rwork (%) / Rfree (%)c

26.0/31.1 (31.1/36.8)

23.7/28.4 (34.5/39.2)

B-factors (Å2)


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Protein

94.2

56.4

Water

-

47.5

0.46

0.28

Bond lengths (Å)

0.016

0.021

Bond angles (°)

2.102

2.175

DPI (Å)d Rms

deviations

from

ideal values

a

Values in parentheses are for the highest-resolution shell.

b

Rmerge = ∑|Ihkl — |/∑ Ihkl, where Ihkl is an intensity measurement for reflection with

indices hkl and is the mean intensity for multiply recorded reflections. c

Free R-value was calculated using 5% of the total reflections chosen randomly and

omitted from refinement. d

Diffraction precision index.47

ATPase Activity. The efficiency of ATP hydrolysis activity of LipB-NBD and the H525A variant was assessed with colorimetric assay to quantify released inorganic phosphate. To assay the ATPase activity as a function of ATP concentration, 4 µM of LipB-NBD or 8 µM of the H525A variant was dissolved in 100 µL of 100 mM HEPES pH 7.5 containing 20% glycerol and 1 mM MgCl2, and the enzymatic reaction was initiated by adding 0-500 µM ATP. After incubation at 25°C for 10 min, and the reaction was terminated by adding 1 mL of BIOMOL® Green Reagent (Enzo Life Sciences, Carlsbad, CA). After a 30 min colorimetric reaction, the absorption at 620 nm (OD620) was measured. Absolute quantification of free phosphate was defined using the standard 11 ACS Paragon Plus Environment

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curve prepared by the measurement of different concentrations of inorganic phosphate. The experiment was carried out at least three times. For the ATPase activity measured with various concentrations of ATP, data points were fitted to the following Hill equation: ‫=ݒ‬

‫ݒ‬୫ୟ୶ ∙ [S]௛ ௛ ‫ܭ‬଴.ହ + [S]௛

where v represents the measured velocity, vmax represents the maximal velocity of reaction when all the enzyme is saturated by substrate, [S] denotes the substrate concentration, K0.5 is the substrate concentration which reaches half of the vmax, and h is the hill coefficient. Circular Dichroism (CD) Spectra. The far-UV (200-260 nm) CD spectra of LipB-NBD and its H525A variant were measured at 25°C on a J-725 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan). The protein was dissolved in 10 mM Tris-HCl pH 7.0. The protein concentration was 0.1 mg/ml and a cell with an optical path length of 2 mm was used. The mean residue ellipticity, θ, expressed in deg cm2/dmol, was calculated using an average amino acid molecular mass of 110 Da. The experiment was carried out twice. Gel-Filtration. LipB-NBD and H525A variant (1 mg/ml) were incubated in 10 mM HEPES pH 7.5 containing 20% glycerol and 1 mM ATP for 1 hour at 20°C. To assess the oligomeric state of the proteins, the samples were loaded onto a Hi-Load 16/60 Superdex 200 pg column (GE Healthcare) equilibrated with the same buffer, and were eluted at 4°C with the solution using a flow-rate of 500 µl/min. The elution was monitored with the absorption profiles recorded at 280 nm. The molecular mass corresponding to an observed peak was deduced from the retention volumes of the 12 ACS Paragon Plus Environment

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Biochemistry

molecules in a gel filtration standard (BIO-RAD, California, USA), consisting of γ-globulin, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa; and vitamin B12, 1.35 kDa. The experiment was carried out twice. Structure Modeling. Structural models for LipD and LipB were constructed by using TolC (PDB: 1EK9) and PrtD from Aquifex aeolicus (PDB: 5L22) as a template, respectively, transmembrane

with region

SWISS-MODEL of

(https://swissmodel.expasy.org/).

LipB

was

predicted

by

using

The SOSUI

(http://harrier.nagahama-i-bio.ac.jp/sosui/).

RESULTS Structure of LipC-. The structure of LipC- was determined at 2.9 Å resolution (Figure 1a). The asymmetric unit of the crystal consists of two molecules (chains A and B). Although the two molecules have an extensive dimer interface, they pack upside down, suggesting that the dimer is a non-natural assembly (see Discussion). Although LipChas 422 residues (Met1-Arg25 and Leu47-Glu443), chain A contains 267 of 422 residues (Asn62-Ser327) and chain B contains 213 of 422 residues (Asn76-Thr293 except for Asp83-Lys86 and Ser95). Both the N-terminal and C-terminal regions of LipC are missing probably due to structural disorder. The structures of two molecules are virtually identical with each other with a root-mean-square deviation (RMSD) value of 0.72 Å for 213 Cα atoms from Asn76-Thr293. We used the structure of chain A to describe the structure of LipC- and further analysis. The overall structure of LipC- monomer is shown in Figure 1b. The structure consists of an α-helical domain (Val97-Thr293) and a lipoyl domain (Asn62-Gln96 and 13 ACS Paragon Plus Environment

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Ala294-Ser327). The overall structures of these domains in LipC- are similar with those in HlyD (PDB: 5C22)37, as shown in Figure 1c. The difference between them is the spacial arrangement of two domains, suggesting a flexible connection between the α-helical domain and the lipoyl domain. The α-helical domain has one short helix, two long helices and two connecting loops (Figure 1d). The short helix, α1 (Val97-Asp125), is linked with one of the long helices, α2 (Pro143-Asp211), through the loop, L1. α2 is linked with the other long helix, α3 (Arg216-Thr293), through the loop, L2. α1 is sandwiched in between α2 and α3. Three helices form a coiled-coil structure, which is 121 Å long. The overall structure of the α-helical domain in LipC- is relatively similar to that in HlyD37 with an RMSD value of 4.97 Å over 196 Cα atoms, as shown in Figure 1e. The α-helical domain in LipC- is slightly longer than that in HlyD (115 Å). The lipoyl domain folds into a lipoyl motif, consisting of four β-strands (Figure 1f). β1 (Lys75-Ile77) and β2 (Val92-Leu94) form an anti-parallel β-sheet, and β3 (Thr302-Val304) and β4 (Asp323-Val325) form an anti-parallel β-sheet. The two sheets pack against each other, forming a β-sandwich. The fold of the lipoyl domain in LipCis highly similar to that in HlyD37 with 71 Cα atoms from Gly61-Val97 to Ala294-Ser327 showing an RMSD value of 1.09 Å (Figure 1g).


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Biochemistry

Figure 1. Structure of LipC-. (a) Dimer formation with chains A and B in the asymmetric unit. (b) Monomer of chain A with α-helical and lipoyl domains. (c) Superposition of α-helical and lipoyl domains of LipC- (green) and HlyD (cyan). (d) α-helical domain with α1 (dark-green), L1 (orange), α2 (light-green), L2 (orange) and α3 (green). N and C represent the N- and C-terminal regions, respectively. (e) Superposition of α-helical domain of LipC- (green) and HlyD (cyan). (f) Lipoyl domain 15 ACS Paragon Plus Environment

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with β1 (blue), β2 (purple), β3 (light-pink) and β4 (pink). N and C represent the N- and C-terminal regions, respectively. (g) Superposition of lipoyl domain of LipC- (green) and HlyD (cyan).

Structure of LipB-NBD. To examine whether the structure of LipB-NBD is similar to that of other NBDs of T1SS,36 the crystal structure of LipB-NBD was determined at 2.65 Å resolution. The asymmetric unit consists of two molecules (chains A and B), which is probably due to artifact of the crystal packing. The structures of chains A and B are well superimposed with a RMSD value of 0.75 Å for 223 Cα atoms. We used the structure of chain A in this study. The overall structure of LipB-NBD is shown in Figure 2, in comparison with the structure of HlyB-NBD,36 which shows 36% amino acid sequence identity to LipB-NBD. The structure of LipB-NBD resembles that of HlyB-NBD with a RMSD value of 2.96 Å for 202 Cα atoms. Notably, the steric configurations of the phosphate binding region or Walker A (Gly365-Thr373), the Mg-binding site or Walker B (Leu488-Glu493), the D-loop (Ser496-Asp499) that includes a coordinating residue of the attacking water, and the H-loop (Ile523-Thr527). It has been reported that His662 of HlyB-NBD is one of the critical residue for ATP hydrolysis activity and ATP-dependent NBD dimerization.48-51 The steric configuration of His525, which is located at H-loop of LipB-NBD, is nearly identical to that of His662 of HlyB-NBD (Figure 2b). His525 of LipB-NBD is well conserved in other NBDs of T1SS, and is probably the key region for ATP hydrolysis of HlyB-NBD.


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Figure 2. Structure of LipB-NBD. (a) Monomer of chain A with Walker A (red), Walker B (purple), D-loop (blue) and H-loop (yellow). N and C represent the N- and C-terminal regions, respectively. (b) Superposition of LipB-NBD (cyan) and HlyB-NBD (gray). The active-site residue His525 of LipB-NBD and the corresponding residue His662 of HlyB-NBD are shown by stick models. N and C represent the N- and C-terminal regions, respectively.

Characterization of LipB-NBD. ATP hydrolysis activity of LipB-NBD was positively correlated with ATP concentration (Figure 3b). In addition, the activity exhibited ATP-dependent positive cooperativity with the Hill coefficient of 1.6, vmax of 563 nmol Pi/min/µmol and K0.5 of 41 µM. It has been well known that Walker A and Walker B are responsible for nucleotide and Mg2+ binding in ABC transporter NBDs.52 To examine whether His525 of LipB-NBD contributes to the ATP hydrolysis and NBD dimerization, 17 ACS Paragon Plus Environment

Biochemistry

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H525A variant was constructed. The variant was purified to give a single band on SDS-PAGE as was LipB-NBD. Figure 3a shows far-UV CD spectra of LipB-NBD and H525A variant measured at pH 7.0 and 25°C. The spectrum of H525A variant was nearly identical to that of LipB-NBD, indicating that the substitution at His525 did not significantly affect the structure of LipB-NBD. As shown in Figure 3b, ATP hydrolysis activity of LipB-NBD was abolished by the His-to-Ala substitution regardless of the addition of ATP, suggesting the importance of His525 in ATPase activity. Analytical size exclusion chromatography showed that LipB-NBD (31.5 kDa) incubated with 1 mM ATP was eluted mainly as a monomer, while H525A variant with 1 mM ATP was mainly as a dimer (Figure 3c). These results indicate that His525 plays an important role in the association and dissociation of a nucleotide-bound dimer. In Figure 3c, there are minor peaks in LipB-NBD at around 100 kDa and in H525A at 30 kDa. After re-chromatography of both major peaks, similar results were obtained. Because SDS-PAGE analysis showed that they were not contaminants (data not shown), there are oligomer-monomer (minor-major) and dimer-monomer (major-minor) equilibrium states in LipB-NBD and H525A, respectively.


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Figure 3. Characterization of LipB-NBD and its active site variant, H525A. (a) Far-UV CD spectra of LipB-NBD (solid line) and H525A (dashed line). (b) The ATP concentration dependence of ATPase activity of LipB-NBD (filled circle) and H525A (filled triangle). (c) Analytical size-exclusion chromatography of LipB-NBD (solid line) and H525A (dashed line) after incubation with 1 mM ATP for 1 hour. Elution volume of the molecular mass (MM) standards is shown.


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Model Structures of LipB and LipD. LipB-NBD forms a dimer in the presence of ATP and forms a monomer in the absence of ATP, as revealed by the analysis of its active site variant, H525A, suggesting that LipB also has a dimer form in inner membrane like general ABC transporters. PrtD from Aquifex aeolicus is an ABC transporter of protease secretion system. Recently, the crystal structure of PrtD has been determined.38 The PrtD structure shows that PrtD forms a dimer with transmembrane domain (TMD) and NBD. LipB is homologous to PrtD with amino acid sequence similarities of 44% in the all regions and 42% in the TMD. Figure 4a shows the model structure of LipB from the crystal structure of PrtD. The fold of LipB is highly similar to that in PrtD with an RMSD value of 0.11 Å. The model structure of the transmembrane region in LipB has six helices. The structure of LipD was predicted from TolC, as shown in Figure 4b. The overall monomer structure of LipD is similar to that of TolC with an RMSD value of 0.68 Å, and the overall trimeric structure of LipD is apparently similar to that of TolC, whereas the RMSD value between them is relatively larger, 13.8 Å, due to a gap through the overall structure of the large molecules.


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Figure 4. Model structure of LipB and LipD. (a) The model dimer structure of LipB from the crystal structure of PrtD from Aquifex aeolicus (PDB: 5L22). (b) The model trimer structure of LipD from the crystal structure of E. coli TolC (PDB: 1EK9).

DISCUSSION LipC. The crystal structure of the α-helical domain and lipoyl domain in LipC- is similar to that in HlyD,37 as shown in Figure 1c. This means that the overall conformations of these domains are commonly conserved in the MFP. However, although LipA is secreted by the LipB-LipC-LipD transporter reconstituted in E. coli, it is not secreted by the LipB-LipC-TolC system.17 The sequence similarity between LipD and TolC (27%) is higher than that between LipC and HlyD (23%). TolC plays a common role in the secretion of diverse molecules, including protein toxins and antibacterial drugs.4,6,8,9 These results suggest that the interaction between LipC and LipD is different from that between HlyD and TolC due to mainly the difference in the structural properties between LipC and HlyD. 21 ACS Paragon Plus Environment

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AcrB-AcrA-TolC (IMP-MFP-OMP) assembly forms the multidrug efflux pump.53-55 The MFP in the system, AcrA, interacts with TolC through its α-helical tip region, which corresponds with the region around L2 in HlyD. In particular, the RLS motif (Arg128/Leu132/Ser139) located at the α-helical tip region is responsible for TolC binding.56-59 In HlyD, Leu (Leu243) is conserved, while Arg and Ser are replaced by Asp (Asp239) and Ala (Ala250), respectively.37 Mutational analysis on this region of HlyD showed that these residues play a crucial role in the secretion of HlyA, as the RLS motif of AcrA. In LipC, the residues that correspond to the RLS motif are Ser204, Leu208 and Pro215 (Figure 5a). Leu is conserved, while Arg and Ser are replaced by Ser and Pro, respectively. Because of non-interaction of LipC with TolC, Ser204 and Pro215 in LipC may inhibit the formation of LipC-TolC complex. Alternatively, TolC-like protein sequence changes may affect the MFP-OMP specific interactions. In any case, it is necessay to resolve the MFP-OMP interaction and its complex form. MacB-MacA-TolC and EmrB-EmrA-TolC assemblies are the macrolide-specific pump and the multidrug efflux pump, respectively, and MacA and EmrA are MFPs that interact with TolC, similar to HlyD and AcrA.60,61 However, the α-helical domains in HlyD and EmrA, which are 115 and 127 Å long, respectively,37,62 are longer than those in AcrA and MacA, which are 60 and 70 Å long, respectively.59,63 The length of the α-helical domain in LipC is comparable with that in HlyD and EmrA. The difference between HlyD/EmrA/LipC and AcrA/MacA is based on the difference in the structures of their IMPs. The structures of AcrB and MacB protrude the periplasm, but HlyB and EmrB have a small periplasmic domain. Prediction by SOSUI showed that the periplasmic region of LipB is only 3.7% (22 residues). The lack of a periplasmic domain of the IMP is related to the long α-helical domain of MFP. 22 ACS Paragon Plus Environment

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The exact function of the lipoyl domain is still unknown, but it was suggested that it may function as a stabilizing factor for the complex assembly.64,65 The lipoyl domain in LipC- is highly similar to that in HlyD. However, the angle formed by the lipoyl and the α-helical domains is different between LipC- and HlyD, which is due to angular and rotational flexibility between the adjacent domains. The opposite side of the α-helical domain on the lipoyl domain could not be observed in the crystal structure of LipC-, indicating the structural flexibility of this region, probably a β-barrel domain, based on the structures of MFPs of MDR.66,67 Because the α-helical and putative β-barrel domains could interact with LipD and LipB, respectively, the lipoyl domain may be a central shaft of LipC while at the complex assembly, substrate binding, or pump cycle with large conformational changes of the α-helical and putative β-barrel domains.8,66 For the structural determination of putative β-barrel domain, it is probably necessary to form a complex with LipB or with inner membrane through the membrane anchor region of LipC. LipC- forms a dimer in solution (see Expression and Purification in Materials and Methods), and the crystal packing shows an upside-down dimer. If MFP makes a tube-like structure for pumping passenger proteins in the periplasm, the subunits probably assemble in a parallel direction. Therefore, the dimer form in the crystals may represent a non-natural assembly. AcrA and MacA form a barrel-like hexameric assembly.60,68

In

AcrB-AcrA-TolC,

each

oligomeric

state

is

3-6-3

(trimer-hexamer-trimer). In contrast, MacB-MacA-TolC has a 2-6-3 complex, because MacB is a dimeric ABC transporter. Similar to MacB, LipB is an ABC transporter, so the LipB-LipC-LipD assembly may assume a 2-6-3 model. The reasons that LipCcould not form a hexamer in solution or crystal might be that the hexameric formation 23 ACS Paragon Plus Environment

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needs the membrane anchor region, the interaction with OMP or IMP, or the interaction with the substrates. For HlyD, a hexameric doughnut-like model was predicted.37 Arg186 and Asp309 of HlyD produce an electrostatic inter-subunit interaction in the predicted hexameric model. The corresponding residues of LipC are Leu151 and Met274 (Figure 5b). LipC may have hydrophobic interactions between them for binding a HlyD-like hexamer, or form a different oligomeric state against HlyD. Leu165, Val334 and Val349 of HlyD affect the secretion and folding of HlyA.11 The corresponding residues of LipC are Ile129, Val299 and Val314, indicating the conservation of these residues. This suggests that LipC forms a hexameric assembly during the transport of passenger proteins as HlyD. The contribution of these residues of LipC to the secretion and folding of a passenger protein will be investigated by the co-expression of PML with Lip systems that contain LipC variants reconstituted in E. coli.


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Figure 5. Structural comparison of LipC and HlyD. (a) Around the RLS motif of LipC (green) and HlyD (cyan). The RLS motif located at the α-helical tip region is responsible for TolC binding in HlyD. The residues corresponding the RLS motif of LipC (yellow) and HlyD (blue) are shown. Leu is conserved, while Arg and Ser in HlyD are replaced by Ser and Pro in LipC, respectively. (b) The modeled interaction between two adjacent protomers of HlyD hexamer (cyan). The electrostatic inter-subunit interaction between Arg186 and Asp309 is shown in blue. The corresponding residues of LipC (green) are Leu151 and Met274 and shown in yellow.

LipB-NBD. ABC transporters share the similar architecture, comprising of TMD and NBD, with a dimer form. Especially, NBDs are more conserved than TMDs in both primary and tertiary structures. The structure of LipB-NBD is similar with those of 25 ACS Paragon Plus Environment

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HlyB-NBD and PrtD-NBD. It is widely accepted that NBDs form a composite dimer in the ATP-bound state, where ATP acts as a molecular glue between NBDs.69,70 In the case of LipB-NBD, the H525A variant without ATP hydrolysis activity forms a dimer in the presence of 1 mM ATP, whereas LipB-NBD does not, resulted from ATP hydrolysis. These results indicate that LipB-NBD has the common overall architecture and ATP hydrolysis activity of ABC transporter NBDs. LipB-LipC-LipD Assembly. Compared with MDR, the complex form of T1SS is still unclear. Here, we predict an assembly of the Lip system using the crystal and predicted structures together with information from previous studies of T1SS6,8,9,64,68,71-74 and MDR.59,75,76 Figure 6 shows a schematic representation of the assembled models of Lip system,64 which is our hypothesis. In the membrane, the ATPase activity of an IMP, macrolide transporter MacB, is stimulated by a MFP, MacA,75 suggesting IMP-MFP complex formation before the substrate binding. The recruitment of TolC by HlyB-HlyD complex is promoted by the substrate binding.71,73 HlyD lacking the N-terminal cytoplasmic helix failes to recruit TolC and activate HlyB-NBD.72 Therefore, in the absence of a substrate, LipB may form a complex with LipC8,9 in an open conformation, resulting in no interaction with LipD. After substrate binding, LipC changes the conformation59,76 and forms a tube-like structure where the α-helical domains surround each other around the lipoyl domains. The tube-like structure is able to interact with LipD, pumping out the substrate.71-74 These conformational changes and the transport process are driven by NBD-mediated ATP hydrolysis. Needless to say, such assembled models remain a matter of our speculation. Further investigations based on the structural information of T1SS could reveal and confirm the transportation


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mechanism of T1SS, which may contribute to biotechnological and pharmaceutical applications.


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Figure 6. Model assembly of Lip system. Left and right figures show the open and closed forms of LipC, respectively. LipB and LipD are represented by a dimer and trimer forms, respectively, but LipC is represented by only two molecules.


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ASSOCIATED CONTENT Accession Codes Coordinates and structure factors have been deposited in the Protein Data Bank as entries 5NEN (LipC-) and 5X7K (LipB-NBD).

AUTHOR INFORMATION Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Dr. K. Omori for the kind gift of plasmid pYBCD20, and H. Nakamura and A. Maeda for the technical support. The synchrotron radiation experiments were performed at SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposals 2016A2572 and 2016B6639).

ABBREVIATIONS ATP, adenosine triphosphate; CAPS, 3-[cyclohexylamino]-1-propanesulfonic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MD, molecular dynamics; tris; tris(hydroxymethyl)aminomethane.


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(28) Amada, K., Kwon, H. J., Haruki, M., Morikawa, M., and Kanaya, S. (2001) Ca2+-induced folding of a family I.3 lipase with repetitive Ca2+ binding motifs at the C-terminus. FEBS Lett. 509, 17-21. (29) Angkawidjaja, C., Paul, A., Koga, Y., Takano, K., and Kanaya, S. (2005) Importance of a repetitive nine-residue sequence motif for intracellular stability and functional structure of a family I.3 lipase. FEBS Lett. 579, 4707-4712. (30) Angkawidjaja, C., Kuwahara, K., Omori, K., Koga, Y., Takano, K., and Kanaya, S. (2006) Extracellular secretion of Escherichia coli alkaline phosphatase with a C-terminal tag by type I secretion system: purification and biochemical characterization. Protein Eng. Des. Sel. 19, 337-343. (31) Kuwahara, K., Angkawidjaja, C., Matsumura, H., Koga, Y., Takano, K., and Kanaya, S. (2008) Importance of the Ca2+-binding sites in the N-catalytic domain of a family I.3 lipase for activity and stability. Protein Eng. Des. Sel. 21, 737-744. (32) Angkawidjaja, C., Matsumura, H., Koga, Y., Takano, K., and Kanaya, S. (2010) X-ray crystallographic and MD simulation studies on the mechanism of interfacial activation of a family I.3 lipase with two lids. J. Mol. Biol. 400, 82-95. (33) Kuwahara, K., Angkawidjaja, C., Koga, Y., Takano, K., and Kanaya, S. (2011) Importance of an extreme C-terminal motif of a family I.3 lipase for stability. Protein Eng. Des. Sel. 24, 411-418. (34) Cheng, M., Angkawidjaja, C., Koga, Y., and Kanaya, S. (2012) Requirement of lid2 for interfacial activation of a family I.3 lipase with unique two lid structures. FEBS J. 279, 3727-3737. (35) Cheng, M., Angkawidjaja, C., Koga, Y., and Kanaya, S. (2014) Calcium-independent opening of lid1 of a family I.3 lipase by a single Asp to Arg mutation at the calcium-binding site. Protein Eng. Des. Sel. 27, 169-176. (36) Schmitt, L., Benabdelhak, H., Blight, M. A., Holland, I. B., and Stubbs, M. T. (2003) Crystal structure of the nucleotide-binding domain of the ABC-transporter haemolysin B: identification of a variable region within ABC helical domains. J. Mol. Biol. 330, 333-342. (37) Kim, J. S., Song, S., Lee, M., Lee, S., Lee, K., and Ha, N. C. (2016) Crystal structure of a soluble fragment of the membrane fusion protein HlyD in a type I secretion system of Gram-negative bacteria. Structure 24, 477-485. (38) Morgan, J. L., Acheson, J. F., and Zimmer, J. (2017) Structure of a type-1 secretion system ABC transporter. Structure 25, 522-529. (39) Goodwin, T. W., and Morton, R. A. (1946) The spectrophotometric determination of tyrosine and tryptophan in proteins. Biochem J. 40, 628-632. (40) Otwinowski, Z., and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307-326. (41) Kabsch, W. (2010) XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125-132.


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Structural Basis for the Serratia marcescens Lipase ... - ACS Publications

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