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Article
Conformational dynamics, intramolecular domain conformation signaling and activation of apo-FimD revealed by single-molecule FRET studies Yanqing Liu, Chuanqi Sun, Long Han, Yuqi Yu, Haizhen Zhou, Qiang Shao, Jizhong Lou, Yongfang Zhao, and Yihua Huang Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00080 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 23, 2019
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Biochemistry
Conformational dynamics, intramolecular domain conformation signaling and activation of apo-FimD revealed by single-molecule FRET studies
Yanqing Liu1,2,#, Chuanqi Sun1,2,#, Long Han1,2, Yuqi Yu3, Haizhen Zhou1, Qiang Shao3, Jizhong Lou2,4, Yongfang Zhao1,2,*,ξ and Yihua Huang1,2,*
1National
Laboratory
of
Biomacromolecules,
CAS
Center
for
Excellence
in
Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China. 2University 3Drug
of Chinese Academy of Sciences, Beijing100101, China.
Discovery and Design Center, CAS Key Laboratory of Receptor Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai, 201203, China. 4Key
Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences,
Beijing100101, China. *Correspondence
should be address to Y.H. (
[email protected]) or Y.Z.
(
[email protected]). # These ξ
authors contributed equally to the work.
Deceased.
Contact phone number: 86-10-64888789
Keywords: the chaperone/usher pathway, outer membrane protein, type 1 pili, single-molecule fluorescence resonance energy transfer (smFRET)
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Abstract The chaperone-usher (CU) secretion pathway is a conserved bacterial protein secretion system dedicated to the biogenesis of adhesive fibers. Usher, a multi-domain containing outer-membrane (OM) protein, plays a central role in this process by acting as a molecular machine
that
recruits
different
chaperone-subunit
complexes,
catalyzes
subunit
polymerization, and forms a channel for secretion of the assembled subunits. While recent crystal structural studies have greatly advanced our understanding of the structure and function of ushers, the overall architecture of the full-length apo-usher, the molecular events that dictate conformational changes of usher during pilus biogenesis and its activation by the specific chaperone-adhesin complex remain largely elusive. Using single-molecule fluorescence resonance energy transfer (smFRET) studies, we found that the substrate-free usher FimD (apo-FimD) adopts a contracted conformation that is distinct from its substrate-bound states; both the N-terminal domain (NTD) and the C-terminal domain (CTD) of apo-FimD are highly dynamic; and FimD coordinates its domain conformational changes via intramolecular domain conformation signaling. Combined with in vitro photocrosslinking studies, we further show that only the chaperone-bound adhesin (FimC-FimH) is able to be transferred to the CTD, dislocates the plug domain, and triggers conformational changes of the rest FimD domains. Taken together, these studies delineate an overall architecture of the full-length apo-FimD, provide a detailed mechanic insight into the activation of apo-FimD, and explain why FimD could adjust its conformational states to perform multiple functions in each cycle of pilus subunit addition and ensure pilus assembly to proceed progressively in a cellular energy-free environment.
1
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Biochemistry
Introduction Pili or fimbriae, a type of adhesive fibers anchored to the surface of many uropathogenic Gram-negative bacteria such as Escherichia coli, are assembled by the conserved chaperone/usher (CU) secretion pathway [1-4]. Type 1 and P pili are prototypical bacterial fibers playing important roles in mediating adhesion of bacteria to the urinary tract in humans [5, 6]. For instance, uropathogenic bacteria use type 1 pili to recognize mannosylated proteins on the bladder epithelium, the first step in the establishment of bacterial invasion and the development of cystitis [6]. The assembly of type 1 pilus takes place at the outer membrane (OM)-periplasm interface and is carried out by two non-subunit component proteins, the chaperone FimC and the usher FimD (Figure S1a). In periplasm, chaperone FimC stabilizes pilus subunits by forming various binary complexes via a mechanism termed donor-strand complementation (DSC) [7, 8]. Subsequently, usher FimD recruits different chaperone-subunit complexes from the periplasm, catalyzes subunit polymerization, and facilitates secretion of the assembled fiber across the OM. The subunit polymerization involves the exchange of chaperone-subunit for subunit-subunit interactions by a mechanism termed donor-strand exchange (DSE) [9, 10]. The assembled type 1 pilus adopts a composite architecture consisting of a rigid helical rod and a short flexible tip that is mounted on the rod [11]. The rod structure contains more than 1,000 copies of FimA, the major pilus subunit; the short tip only consists of three pilus subunits, with the adhesin subunit FimH located at its distal end, followed by single copies of FimG and FimF [12, 13] (Figure S1a). FimD, a large integral OM protein, plays a central role in the biogenesis of type 1 pilus. Schematically, FimD can be divided into five distinct domains: a periplasmic N-terminal domain (NTD), a transmembrane β-barrel, a β-sandwich plug domain that is located within the β-barrel region, and two periplasmic C-terminal domains (CTD), CTD1 and CTD2 [14-18] (Fig. 1a). The NTD is responsible for recruiting different chaperone-subunit complexes in the periplasm [16, 19-21]; the CTD is crucial for handover of the recruited chaperone-subunit complex from the NTD to the CTD and anchoring the growing fiber by providing a second higher-affinity binding site [2, 19, 22, 23]; the transmembrane β-barrel 2
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functions as a secretion channel for the polymerized subunits, but it remains sealed by the plug domain in the resting state to prevent toxic substances from entry into the periplasm [15, 17, 18]. Although in vitro studies show that the NTD is able to bind different chaperone-subunit complexes with differential affinities, the assembly of type 1 pilus proceeds in an ordered manner, primed by the chaperone-bound adhesin subunit FimH, and followed by addition of FimG, FimF and FimA sequentially both in vivo and in vitro [24-27]. Despite crystal structures available for the chaperone-subunit bound FimD and the isolated usher domains [15, 17-19, 21, 22, 28, 29], the overall architecture of the full-length apo-usher and molecular events that usher coordinates the conformation of its multiple domains during pilus biogenesis and its activation by the specific FimC-FimH complex remain obscure . In this report, using single-molecule fluorescence resonance energy transfer (smFRET) study, we depicted the overall architecture of the full-length apo-FimD (the inactive state) that is missing in previous extensive structural studies, and found that apo-FimD adopts a contracted conformation with its NTD and CTD in a close distance to each other as well as to its β-barrel domain, in sharp contrast to its extended conformation that is observed in its substrate-bound states [18, 28]. We also observed that both the NTD and the CTD of apo-FimD are highly dynamic, and FimD coordinates its domain conformation via intramolecular domain conformation signaling. Combined with in vitro photocrosslinking, we further show that only the FimC-FimH complex is able to be transferred to the CTD of apo-FimD and has the capacity to displace the plug domain from the β-barrel lumen, thus activating apo-FimD. Materials and methods Preparation of FimD and the elongation complex (FimD:FimC:FimF:FimG:FimH) for smFRET experiments. The gene encoding the full-length FimD, FimC, FimF, FimG, FimH including their natural signal sequences were amplified from Escherichia coli O6:H1 (strain CFT073 / ATCC 700928 / UPEC) genomic DNA by PCR. The five genes were individually inserted into the pQLink expression vector via BamHI/HindIII restriction sites to construct plasmids pQLink-FimD, pQLink-FimC, pQLink-FimG, pQLink-FimF and pQLink-FimH. To facilitate 3
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Biochemistry
affinity purification, a His6-tag was attached to the C-terminus of FimD. For co-expression of the elongation complex, the five plasmids were further combined step-by-step by ligation-independent pathway to generate plasmid pQLink-FimDCHGF, which allows co-expression of the five proteins in one plasmid. All the amber codon mutations and domain deletion mutations were carried out using either pQLink-FimD or pQLink-FimDCHGF as templates. For protein expression, both plasmids pEVOL-pAzF (Addgene) and pQLink-FimD (or pQLink-FimDCHGF) that contains double amber stop codon (TAG) mutation at selected positions in FimD sequence were co-transformed into E. coli strain C43 (DE3). The plasmid pEVOL-pAzF
encodes
an
L-arabinose-inducible
amber
suppressor
tRNA
and
aminoacyl-tRNA synthetase, allowing incorporation of pAzF at amber stop codons in FimD sequence. pAzF was added to a final concentration of 1 mM in LB when cells grew to mid-log phase (OD600 = 0.2-0.3). Protein expression was induced at OD600 of 0.8 with 0.02% L-arabinose and 100 μM isopropyl-β-D-thiogalactopyranoside (IPTG) for 15 h at 25℃. Cultures were harvested and subsequently resuspended in 20 ml phosphate-buffered saline (PBS) pH 7.4, followed by sonication through a French Press (JN-3000 PLUS, China) at
16,000
p.s.i.
The
cell
membranes
were
solubilized
with
1%
(w/v)
n-dodecyl-β-D-maltopyranoside (DDM; Anatrace) for 1 h at 4 ℃. Solubilized membranes were loaded onto a Ni-NTA superflow affinity column equilibrated with a buffer containing 50 mM HEPES (pH 7.0), 150 mM NaCl and 0.05% (w/v) DDM. FimD or the elongation complex proteins were eluted using the same buffer containing 250 mM imidazole. In order to obtain homogeneous elongation complex, the elongation complex samples were further passed through an α-D-mannose-agarose column (Sigma-Aldrich) as previously described [30]. Protein labeling and steady-state fluorescence anisotropy measurements. For smFRET experiments, protein samples (2 µM) were labeled in the buffer (50 mM HEPES [pH 7.0], 150 mM NaCl and 0.05% [w/v] DDM) containing a mixture of 20 μM DBCO-Sulfo-Cy3 and DBCO-Sulfo-Cy5 dyes (Jena Bioscience) for 2 hrs at room temperature. Free dyes were removed using ZebaTM Spin desalting column (Thermo 4
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Scientific). In addition, we carried out labeling efficiency measurement for the protein that harbors single amber stop codon mutation site with DBCO-Sulfo-Cy3. We estimated the extent of the labeling from absorption spectra of labeled protein by measuring peak maxima at 553 nm using NanoPhotometer P330 (IMPLEN, Germany). The concentration of labeled-protein was measured by Bicinchoninic Acid (BCA) Protein Assay Kit (CWBIO, China). The labeling ratio for DBCO-Cy3 was around 80%. Anisotropy measurements for DBCO-sulfo-Cy3-labelled protein were carried out using F7000 ultraviolet spectrophotometer (Sigmatech Inc.) with excitation and emission wavelengths of 532 nm and 570 nm, respectively. Single-molecule FRET (smFRET) measurements. Imaging channels passivated with a mixture of PEG and biotin-PEG were incubated with 100 µg ml-1 streptavidin. Protein was immobilized to the streptavidin-treated channel surface through Biotin-NTA-Ni2+ (Biotium). All experiments were performed in the buffer containing 50 mM HEPES (pH 7.0), 150 mM NaCl and 0.05% (w/v) DDM with an oxygen scavenging system (0.1% v/v glucose, 5 mM β-mercaptoethanol, 1 unit/µl glucose oxidase, 8 unit/µl catalase, and 1 mM cyclo-octatetraene) [31]. Images were taken at 50 ms per frame. Cy3 and Cy5 channel were mapped using TetraSpeck fluorescent microsphere beads (Invitrogen, 0.1 µm). At least more than 10 beads were selected to obtain the transformation matrix used in mapping in MatLab. Fluorescence data were acquired using an objective based total internal reflection fluorescent (TIRF) microscope and Cy3 fluorophore was excited with 532 nm lasers (Coherent Inc., Sapphire SF). 1.49 NA 100× OTIRF (Olympus UAPON) was used to collect the photon emitted from Cy3 and Cy5, whose frequency signals were separated spatially onto a cooled EMCCD (Andor iXon Ultra) by Optosplit II (Cairn Research Limited). Fluorescence data were acquired using the software Metamorph (Universal Imaging Corporation). Detailed methods of data analysis were introduced in previous studies [32]. Analysis of single-molecule fluorescence data was performed using custom software written in MATLAB (MathWorks). A subset of the acquired traces was selected for further analysis using the following criteria: (1) single-step donor photobleaching, (2) signal-to-background noise ratio 5
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Biochemistry
(SNR) ≥ 8, (3)
