The N-terminal domain of MuB protein has striking structural similarity to DNA-binding domains and mediates MuB filament-filament interactions.

Journal of Structural Biology xxx (2015) xxx–xxx

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Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi

The N-terminal domain of MuB protein has striking structural similarity to DNA-binding domains and mediates MuB filament–filament interactions Marija Dramicánin a,1,3, Blanca López-Méndez b,2,3, Jasminka Boskovic c, Ramón Campos-Olivas b, Santiago Ramón-Maiques a,⇑ a Structural Bases of Genome Integrity Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fdez. Almagro, 3, Madrid 28029, Spain b Spectroscopy and Nuclear Magnetic Resonance Unit, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fdez. Almagro, 3, Madrid 28029, Spain c Electron Microscopy Unit, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fdez. Almagro, 3, Madrid 28029, Spain

a r t i c l e

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Article history: Received 23 May 2015 Received in revised form 8 July 2015 Accepted 9 July 2015 Available online xxxx Keywords: NMR structure Phage Mu Transposition immunity Helix-turn-helix k repressor-like

a b s t r a c t MuB is an ATP-dependent DNA-binding protein that regulates the activity of MuA transposase and delivers the target DNA for transposition of phage Mu. Mechanistic insight into MuB function is limited to its AAA+ ATPase module, which upon ATP binding assembles into helical filaments around the DNA. However, the structure and function of the flexible N-terminal domain (NTD) appended to the AAA+ module remains uncharacterized. Here we report the solution structure of MuB NTD determined by NMR spectroscopy. The structure reveals a compact domain formed by four a-helices connected by short loops, and confirms the presence of a helix-turn-helix motif. High structural similarity and sequence homology with k repressor-like DNA-binding domains suggest a possible role of MuB NTD in DNA binding. We also demonstrate that the NTD directly mediates the ability of MuB to establish filament–filament interactions. These findings lead us to a model in which the NTD interacts with the AAA+ spirals and perhaps also with the DNA bound within the filament, favoring MuB polymerization and filament clustering. We propose that the MuB NTD-dependent filament interactions might be an effective mechanism to bridge distant DNA regions during Mu transposition. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction DNA transposons are mobile genetic elements that translocate from one site to another within the genome. Phage Mu is among the most complex and efficient DNA transposons, and its study

Abbreviations: HTH, helix-turn-helix; NMR, nuclear magnetic resonance; NTD, N-terminal domain; Oct, octamer binding transcription factor; POUS, POU specific DNA-binding domain; POUHD, POU homeodomain; RMSD, root-mean-squared deviation. ⇑ Corresponding author. E-mail address: [email protected] (S. Ramón-Maiques). 1 Present address: ACRF Chemical Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia. 2 Present address: Biophysics Unit, Protein Production and Characterization Platform, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. 3 These authors contributed equally to this work.

set the bases for our current understanding of transposition mechanisms, retroviral integration and early steps of V(D)J recombination (reviewed in (Chaconas and Harshey, 2002; Harshey, 2012; Mizuuchi, 1992)). Efficient Mu transposition requires the interplay between two phage proteins, MuA and MuB, and their assembly into higher-order protein–DNA complexes (Mizuuchi et al., 1995). The transposase MuA recognizes the two ends of the Mu sequence and brings them into proximity forming a stable complex or ‘‘transposome’’. Within this complex, MuA catalyzes the cleavage of the Mu ends and their insertion into a new target DNA site (Craigie and Mizuuchi, 1987; Mizuuchi, 1992; Surette et al., 1987). MuB, on the other hand, is an activator protein, required to favor the assembly of MuA at the transposon ends (Mizuuchi et al., 1995), to stimulate the catalytic activities of MuA (Baker et al., 1991; Surette et al., 1991) and to deliver the target DNA to the transposome (Maxwell et al., 1987). Biochemical and structural studies, including the crystal structure of a MuA tetramer

http://dx.doi.org/10.1016/j.jsb.2015.07.004 1047-8477/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Dramicánin, M., et al. The N-terminal domain of MuB protein has striking structural similarity to DNA-binding domains and mediates MuB filament–filament interactions. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.07.004

M. Dramicánin et al. / Journal of Structural Biology xxx (2015) xxx–xxx

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interlaced with two Mu ends and a sharply curved target DNA (Montano et al., 2012), have allowed for dissection of the architecture and catalytic mechanism of the transposome. However, obtaining structural information about MuB has proven difficult due to its tendency to aggregate in vitro in the presence of ATP. Thus, a detailed description of how MuB carries out its multiple functions during transposition remains elusive. MuB is a small (35 kDa) ATP-dependent DNA-binding protein with relatively low ATPase activity (Maxwell et al., 1987). In the presence of ATP, MuB oligomerizes and binds non-specifically and with high affinity to DNA to make it a preferred target for Mu transposition (Adzuma and Mizuuchi, 1988, 1991; Greene and Mizuuchi, 2002b; Yamauchi and Baker, 1998). At the same time, MuA interacts with MuB stimulating ATP hydrolysis and the dissociation of MuB from the DNA (Adzuma and Mizuuchi, 1988; Maxwell et al., 1987). Thus, by the time MuA assembles an active transposome and is ready to capture the target DNA, MuB has been removed from the neighborhood and accumulates at distant DNA regions. This biased distribution of MuB along the DNA explains the strong preference for the transposition to occur 5– 25 kb away from the initial insertion site (Adzuma and Mizuuchi, 1989; Manna and Higgins, 1999). This phenomenon is called transposition target immunity since it prevents the destructive insertion of the transposon into its own DNA. The dynamics of the interaction between MuB and DNA were first described by total internal reflection fluorescence (TIRF) microscopy using fluorescently labeled MuB and immobilized single duplex DNA molecules (Greene and Mizuuchi, 2002a, 2004). In the ATP-bound form, GFP–MuB binds along the DNA forming many short and separated filaments, and, as more protein is added, the filaments elongate to fully cover the DNA molecule. More recently, mechanistic insight into MuB polymer assembly and a close-up view of the filament structure were obtained. MuB was shown to be composed of an AAA+ (ATPases Associated with diverse cellular Activities) ATPase module (residues 77–312) and an N-terminal appended domain (hereafter called NTD; residues 1–76) (Fig. 1A) (Mizuno et al., 2013). The AAA+ module was shown to be necessary and sufficient for filament formation, as well as for the ATPase activity and for the interaction with DNA and with MuA. Cryo-electron microscopy (EM) was used to obtain 3D reconstructions of the filaments at 18 Å resolution. In the absence of DNA, MuB-ATP was shown to polymerize into a right-handed helical filament with an axial channel of the appropriate dimensions to allocate a double stranded DNA molecule (Fig. 1B) (Mizuno et al., 2013). Indeed, in the presence of DNA, MuB helical filaments wrap around the DNA molecule, and although the protein helical parameters do not match those of the B-form DNA, the structure of the DNA within the filament is seemingly unaltered. On the other hand, the NTD, which was proposed to hold a putative helix-turn-helix (HTH) motif (Miller et al., 1984), appears to be flexibly attached to the AAA+ module (Fig. 1B) and could be removed without severely affecting DNA binding or impeding the known functions of MuB. Indeed, a truncated protein lacking the NTD (MuB-DN) was demonstrated to form similar filaments as MuB wild-type (MuBwt) (Mizuno et al., 2013). However, MuB-DN retains only half of the ATPase activity of MuBwt and has a compromised capacity to stimulate MuA activity and to discriminate against ‘‘immune’’ target DNA (Mizuno et al., 2013). Therefore, the function of the appended NTD remains uncharacterized. In this study, we have determined the solution structure of MuB NTD by NMR spectroscopy. The domain folds into four compact a-helixes with a characteristic HTH motif. Despite the structural similarity with DNA-binding domains of the k repressor-like superfamily, we could not detect a direct interaction between the isolated NTD and the DNA. However, we demonstrate that the NTD

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Temperature (ºC) Fig. 1. NTD purification and preliminary characterization. (A) Schematic representation of MuB protein. (B) 3D reconstruction of MuB helical filament; the axial channel where DNA binds is colored in magenta. The NTD, represented for one MuB subunit, is flexibly attached to the AAA+ module and not seen in the 3D reconstruction. (C) NTD purification monitored by SDS–PAGE. Ni, elution from His-trap column; DD, dialysis and digestion with PreScission protease; Q, ion exchange column; GF, gel-filtration. Molecular-mass standards are indicated on the left. (D) Measurement of NTD molecular mass by multi angle light scattering (MALS) coupled to size exclusion chromatography. (E) Circular dichroism absorption spectrum of the NTD with characteristic a-helical bands at 208 and 220 nm. (F) NTD unfolding measured by changes in ellipticity at 222 nm as a function of temperature. The melting temperature at which half of the protein is unfolded is indicated.



is essential for the interaction between MuB filaments. Based on these results, we propose a model where the NTD binds between the spirals of the AAA+ helices making protein–protein contacts that favor filament bundling, potentially reaching the axial channel where it may also interact with the DNA. We speculate on the possible functions of the NTD in stabilizing MuB–DNA complexes and in bridging distant regions of the DNA. 2. Materials and methods 2.1. Cloning, protein expression and isotope labeling MuB NTD coding sequence (residues 1–63) was amplified by PCR using as primers 50 -AAGTTCTGTTTCAGGGCCCGATGAATATTT CCGATATTCG-30 and 50 -ATGGTCTAGAAAGCTTTATGCATGATATTTT TCCAGC-30 , and cloned into pOPIN-B (Oxford Protein Production Facility UK) digested with KpnI and HindIII using In-Fusion (Clontech). BL21 (DE3) pLysS Escherichia coli cells (Novagen) transformed with the pOPIN-B-MuB NTD plasmid were grown at 37 °C in LB medium supplemented with 50 lg ml1 kanamycin and 34 lg ml1 chloramphenicol to mid-exponential phase (OD600 = 0.6–0.8). Protein expression was induced with 0.8 mM isopropyl-D-thiogalactopyranoside (IPTG) at 37 °C. After 4 h, the cells were harvested, washed with PBS buffer, and stored at 80 °C. Alternatively, protein was expressed by auto-induction (Studier, 2005). For this, an overnight bacterial preculture grown in ZYP medium with 0.8% (w/v) glucose was inoculated in ZYP-5052 media, grown at 37 °C to OD600 = 0.6–0.8, and incubated at 25 °C for 12 h. The auto-induced expression yielded 3 mg of pure NTD per liter of culture and IPTG-induction yielded 1 mg. To label the protein with 15N for NMR studies, the bacteria were grown in M9 minimal medium containing 1 g l1 of 15N ammonium chloride (Sigma) as the nitrogen source, and protein expression was induced with IPTG. 2.2. Protein purification The cells were resuspended in buffer A (20 mM Tris–HCl pH 8, 0.5 M NaCl, 10 mM imidazole, 2 mM b-mercaptoethanol) with 2 mM phenylmethanesulfonyl fluoride (PMSF) and disrupted by sonication. The lysate was clarified and applied to a 5 ml HisTrap HP column (GE Healthcare) equilibrated in buffer A. After washing with 200 ml of buffer A plus 40 mM imidazole, MuB NTD was eluted in buffer A plus 300 mM imidazole. The sample was dialyzed at 4 °C against buffer B (20 mM Tris–HCl pH 8, 75 mM NaCl, 1 mM DTT), adding PreScission protease (1:20, protease-to-protein ratio) within the dialysis bag. Complete cleavage of the N-terminal His6-tag was achieved in 4 h. The sample was applied to a 5 ml HiTrap Q FF column (GE Healthcare) equilibrated in buffer B, and MuB NTD was eluted by a salt gradient at approximately 0.15 M NaCl. The sample was concentrated by centrifugal ultrafiltration (Amicon Ultra of 3 kDa cutoff; Millipore) and loaded onto a Superdex75 (GE Healthcare) gel filtration column equilibrated in buffer GF (20 mM Tris–HCl pH 8, 0.1 M NaCl). Protein was concentrated to 15 mg ml1, flash-frozen in liquid nitrogen and stored at 80 °C. 15 N labeled protein was purified following the same protocol, except for buffer GF that was replaced by 20 mM sodium phosphate pH 7.5, 0.1 M NaCl, and for the addition of 5.5% D2O to the final protein sample. Protein concentration was determined by optical density at 280 nm using the extinction coefficient of 8480 M1 cm1 or by Bradford. MuB wild-type and MuB-DN were produced as previously described (Mizuno et al., 2013).

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2.3. Site-directed mutagenesis Mutagenesis was carried out following the Quick-Change protocol (Stratagene) by using the pair of mutagenic oligonucleotides 50 -GAGAGCGGGCTTTCTACCGAAACTATCAGTAGTTTTATC-30 and 50 -GATAAAACTACTGATAGTTTCGGTAGAAAGCCCGCTCTC-30 with the flanking primers 50 -AAGTTCTGTTTCAGGGCCCGATGAATATTTC CGATATTCG-30 and 50 -ATGGTCTAGAAAGCTTTAATTACGCAGCAGC GTTG-30 . The amplified fragment with the G33E mutation was inserted into pOPIN-F with In-Fusion. The expression and purification of MuB-G33E was performed as described above for MuBwt. MuB NTD G33E mutant was obtained introducing a stop codon after residue A63, as described for NTD cloning, and the mutated gene was cloned in pOPIN-B. 2.4. Gel-filtration chromatography and multi-angle light scattering For molar-mass determination, 500 ll of purified NTD at 0.6 mg ml1 (82 lM) was fractionated by gel filtration on a Superdex 75 10/300 column equilibrated in buffer GF and characterized by in-line measurement of the refractive index and multi-angle light scattering using Optilab T-rEX and DAWN 8+ instruments, respectively (Wyatt). Data analysis was done with ASTRA 6 software (Wyatt) (Wyatt, 1993). 2.5. Circular dichroism and thermal unfolding MuB NTD samples were prepared at 0.07 mg ml1 (10 lM) and the ellipticity was measured at 200–260 nm using a JASCO J-810 spectropolarimeter with a quartz cuvette of 0.1 cm path length. Thermal melts were run at 1 °C min1 monitoring the ellipticity at 222 nm with a bandwidth of 2 nm and a data pitch of 0.2 °C. The thermal range of the experiments was from 20 °C to 99 °C and the recovery of the ellipticity signal was over 98% after thermal unfolding. Thermal denaturation curves were analyzed by a non-linear least squares fitting algorithm using program Origin (OriginLab), and assuming the linear extrapolation method for a two-state unfolding without populated intermediate states. 2.6. Protein solubility test 30 ll samples of MuBwt, MuB-DN or MuB-G33E at 0.5 mg ml1 in 20 mM Tris–HCl pH 8, 0.17 M NaCl and 5 mM MgCl2 were incubated at 4 °C with or without 1 mM ATPcS (Roche) in the presence or absence of a 56 bp double stranded DNA. After 1 h, the samples were centrifuged at 16,000g for 15 min and protein in the supernatant was measured by Bradford. 2.7. MuB filament visualization by negative staining electron microscopy For electron microscopy (EM) visualization, MuBwt, MuB-DN or MuB-G33E were diluted to 0.07 mg ml1 in 30 mM Tris–HCl pH 8, 0.15 M KCl, 5 mM MgCl2, and 1 mM DTT, and incubated with 1 mM ATP or ATPcS from 10 s. to 12 h. To test filament formation on DNA, a DNA PCR fragment of 1 kb was used at 2.4 ng ll1, and the buffer was supplemented with 0.3 M KCl. 5 ll of sample was applied to a glow-discharged carbon-coated copper grid, washed with three drops of deionized water, and stained with two drops of 1% (w/v) uranyl acetate. The grids were viewed with a Tecnai G2 Spirit electron microscope (FEI, Netherlands) equipped with a LaB6 filament and operated at 120 kV. Images were recorded on a TemCam-F416 4 k_x_4k pixel camera (TVIPS GmbH, Gauting, Germany) at a nominal magnification of 61,320.


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2.8. NMR spectroscopy data acquisition and NOESY cross-peak assignment Unlabeled and 15N labeled samples were prepared in 20 mM sodium phosphate pH 7.5, 0.1 M NaCl, and 5.5% (v/v) D2O at 500 lM (3.7 mg ml1) protein concentration. NMR spectra were recorded at 25 °C on a Bruker Avance 700 MHz spectrometer equipped with a room temperature TXI triple resonance probe. Identification of intraresidual spin systems was achieved using a two dimensional (2D) TOCSY spectrum recorded with 20 ms mixing time, a 2D 1H–15N HSQC and a three dimensional (3D) HNHA spectrum (Cavanagh, 2007). This was used to assign the intraresidue alpha protons (1Ha). A 2D NOESY and a 3D 15N-NOESY-HSQC spectra (150 ms mixing times) were used together with a 2D TOCSY (100 ms mixing time) to establish the sequential connectivities and 1H side-chain assignments. The watergate pulse sequence was used for water suppression in all recorded spectra. The carbon chemical shifts were assigned from a natural-abundance 2D 1 H–13C HSQC spectrum measured in D2O buffer. Heteronuclear 1 H}15N nuclear Overhauser effect (XNOE) of backbone amides were measured from the intensity ratio of signals in spectra recorded with proton saturation over those in reference spectra recorded with a recovery delay of 8 s (Vega-Rocha et al., 2007). Errors were determined from the baseline noise, as implemented in NMRViewJ (Johnson, 2004). All spectra were processed with NMRPipe (Delaglio et al., 1995) and visualized and analyzed with the CcpNmr analysis suite (Vranken et al., 2005). 2.9. NMR structure calculation 3D structures were calculated with CYANA (Guntert, 2004; Mumenthaler et al., 1997) on the basis of the sequence specific chemical shift assignments and NOE peak lists generated with CcpNmr. Dihedral u and w backbone torsion angle restraints were obtained with the program TALOS+ (Shen et al., 2009) using 1Ha, 15 N, 13Ca and 13Cb chemical shifts and incorporated in the calculations. Seven cycles of automated NOE assignment and structure calculation were run, followed by a final structure calculation that uses only unambiguous assigned distance restraints. The structure calculation was started in each cycle from 100 conformers with random torsion angle values. The 20 conformers with lowest final CYANA target function were retained for analysis and guided NOE assignment in the next cycle. Assignments with an overall probability below 10% in cycle 1 or 20% in cycles 2–7 were discarded. The 20 conformers with lowest final CYANA target function values were immersed in an 8 Å shell of explicit water molecules and subjected to restrained energy minimization against the AMBER force field (Cornell, 1995) using the program OPALp (Luginbuhl et al., 1996). CYANA was used to obtain all statistics on NOE assignments, conformational restraints, target function values and restraint violations (Table 1). Ramachandran plots were calculated with PROCHECK (Laskowski et al., 1996). Conformational energies were calculated with OPALp. Structures were visualized and represented with PyMOL (Schrödinger, LLC). 2.10. DNA binding assays DNA binding experiments were done with 15N labeled NTD at 50 lM (0.37 mg ml1), prepared in 20 mM sodium phosphate pH 6.0 or sodium acetate pH 5.0, 0.1 M NaCl and 5.5% (v/v) D2O, with a 5-fold molar excess of a 14 bp double stranded DNA (50 -CGTAT GCAAATCGG-30 ). 2D 1H–15N HSQC spectra were acquired at 298 and 283 K. Binding to DNA was also monitored by electrophoretic mobility shift assays (EMSA). [32P]-labeled double stranded DNA molecules of 12 bp (50 -GTATGCAAATGA-30 ), 14 bp (50 -TGTATGCAAATAAG-30

Table 1 Summary of NMR experimental restraints and structural statistics. NOESY cross-peaksa Total 3464 Assigned 3461 2D NOESY 2645 15 816 3D N-NOESY-HSQC Unassigned 3 Distances restraints Total 1518 Short-range |i j| 6 1 696 Medium-range 1 < |i j| < 5 390 Long-range |i j| P 5 432 Angle restraints (TALOS+) u/w 46/46 Structure statisticsb CYANA target function (Å2) 0.71 ± 0.17 AMBER energy (kcal/mol) 2879.94 ± 76.00 Average RMS deviations from restraints Distance restraints (Å) 0.0105 ± 0.0004 Angle restraints (°) 0.1691 ± 0.0591 Average number of violations Distances restraints > 0.20 Å 0 (maximal 0.16 Å) Dihedral restraints > 5° 0 (maximal 2°) Average RMS deviations from ideal covalent geometry Bonds (Å) 0.0141 ± 0.0001 Angles (°) 1.663 ± 0.045 c Ramachandran plot (%) 93.1/6.6/0.3/0.0 RMSD to mean coordinatesd (Å) 0.41/0.86

99.91% 99.88% 100% 0.09%

45.85% 25.69% 28.46%

a NOESY cross-peaks used to generate distance restraints within the automated NOE assignment algorithm in CYANA. b Calculated over the final bundle of 20 representative conformers after energy minimization with OPALp against the AMBER force field. c Percentage of residues in most favored, additionally allowed, generously allowed, and disallowed regions of the Ramachandran plot. d RMSD values for the backbone atoms N, Ca, C0 or for all the heavy atoms, respectively, in the structured region of the protein spanning residues 2–63. To obtain the RMSD value of a structure represented by a bundle of conformers, all conformers are superimposed on the first one and the average of the RMSD values between the individual conformers and their average coordinates are computed.

or 50 -CGTATGCAAATCGG-30 ) or 39 bp (50 -GGCCTTGTGCTGATGGG AGCGCACCGGGTTTATTCAAAT-30 ) at 45 lM were incubated with 1 mM NTD in buffer 20 mM Tris pH 8, 0.1 M NaCl, 5 mM MgCl2 and 1 mM DTT. After 1 h incubation at 4 °C, the 10 ll reactions were mixed with 20% glycerol and separated by electrophoresis in 15% polyacrylamide gels containing 0.5 TBE buffer. The gels were dried on Whatman paper, exposed on a storage phosphor screen and scanned with a Typhoon Trio (GE Healthcare). 3. Results 3.1. The N-terminal appendage of MuB folds as an independent globular domain Sequence analysis with program PSIPRED (Buchan et al., 2013) predicted that MuB residues 1 to 63 fold into four a-helices connected to the AAA+ module by a disordered stretch of 13 amino acids (Fig. 1A). To further study this region of MuB, we engineered a construct consisting of residues 1 to 63 fused to an N-terminal His6-tag removable by digestion with PreScission protease. The protein, isolated without tag and of high purity, migrates at the expected molecular weight of 7.4 kDa in SDS–PAGE (Fig. 1C). This polypeptide includes two additional residues (GP) at the N-terminus, derived from the protease cleavage site. The purified protein, measured by multi-angle static light scattering (MALS) coupled in-line to a gel-filtration column, exhibits a single monodisperse peak with a calculated molecular mass of 7.40 ± 0.02 kDa (Fig. 1D). The protein is stable and highly soluble, reaching concentrations up to 90 mg ml1. Circular dichroism (CD) analysis showed clear negative bands at 208 and 220 nm,



confirming the a-helical content (Fig. 1E). Analysis of the CD signal at 222 nm as a function of temperature was used to monitor the stability of the polypeptide (Fig. 1F). The unfolding curve followed a single sigmoidal transition and the midpoint indicated a melting temperature (TM) of 59 °C. Overall, these results prove that the N-terminal sequence appended to the AAA+ module is a topologically distinct region of MuB, which adopts a stable a-helical fold and behaves as a monomer in solution. 3.2. NMR structure determination The three dimensional structure of MuB NTD was determined by NMR spectroscopy. The NMR samples used for sequential assignment and structure determination comprised both non-labeled and 15N isotopically labeled protein. The assignment of 1H, 15N, and 13C resonances was accomplished using a combination of 2D 1H–1H TOCSY, 2D 1H–1H NOESY, 2D 1H–15N and 1H–13C HSQC, 3D HNHA and 3D 15N-NOESY-HSQC (see details in Materials and Methods). The 2D 1H–15N HSQC spectrum shows a well-dispersed uniform intensity set of backbone amide correlations (Fig. 2A). Overall, 97.6% of backbone amide and non-labile aliphatic side-chain proton resonances were assigned. The backbone amide signals of L26 and D41 overlapped and those of residues T32, G33, G45, D46 and N47 were absent, probably due to rapid exchange with the solvent. However, CaHa and side-chain resonances were clearly assigned for all residues. The N-terminal additional GP residues exhibited no signals suggesting that they are unstructured in solution. Otherwise, the uniform high values from the {1H}15N nuclear Overhauser effect (XNOE) indicated that the overall structure is rigid, with the exception of the N and C-terminal residues and the loop connecting helices h3 and h4 (Fig. 2B). The NMR solution structure was calculated by simulated annealing in torsion angle space. In total, we used 3461 distance restraints from integrated 2D and 3D NOESY cross-peaks and 92 dihedral u and w torsion angle restrains predicted with the program TALOS+ (Shen et al., 2009). The structures have no violations greater than 0.2 Å or 5° for the experimental distances and angle restraints, respectively, and show favorable conformational energies and Ramachandran plot statistics. A summary of the NMR data collection and model statistics is detailed in Table 1. The ensemble of a representative set of 20 NMR structures superimposed on the backbone atoms is shown in Fig. 2C. The precision of the NMR structure, measured by the root-mean-squared deviation (RMSD) value relative to the average coordinates of non-hydrogen atoms from residue N2 to A63, is 0.41 Å for the main chain and 0.86 Å if side-chains are included. The smaller RMSD between the four a-helices (0.29 Å for backbone and 0.78 Å including side chains) and the high XNOE values are consistent with a rigid and well-defined helical protein core, while small variations occur in the flexible loops connecting helices h2, h3 and h4 (Fig. 2B). In summary, the NTD structures are in excellent agreement with the NMR data and prove that the NTD forms a compact well-defined globular domain. 3.3. Structure of MuB NTD MuB NTD structure shows four a-helices packed against one another and connected by short loops, with approximate dimensions 30 Å 25 Å 25 Å (Fig. 2C and Fig. 3A). Helices h1 (residues 3–15) and h4 (residues 47–61) are longer, with 13 and 15 amino acids, respectively, and are disposed in a parallel orientation forming an angle of 50 degrees between their helical axes. The other two helices, h2 (residues 21–28) and h3 (residues 32–40), are shorter and connected by a 3 residue loop that makes a sharp turn

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mediated by residue G29 (/ = 84 ± 9°, w = 2 ± 7°). As correctly predicted (Miller et al., 1984), the nearly perpendicular arrangement of helices h2 and h3 corresponds to a characteristic HTH motif, a structure typically involved in DNA recognition and integrated in many different prokaryotic and eukaryotic DNA binding proteins (Rohs et al., 2010). The cluster of the four a-helices encloses a hydrophobic core formed by the side chains of residues I3, I6, L10, L13 and V14 from helix h1, F21 and I24 from helix h2, L30 from the loop h2–h3, I35, F38 and I39 from helix h3, and V50, L54, W57 and L58 from helix h4 (Fig. 3A). Only one hydrophobic residue, L26 in helix h2, is not in contact with the hydrophobic core and is exposed to solvent. The rest of the surface residues in helices h1, h2 and h4 have a hydrophilic character, with abundance of charged residues (Fig. 3A). The solvent exposed region of helix h3 is particular due to the presence of the small residues T32, G33, S36 and S37. These residues provide a relatively flat surface roughly delimited by the side chain of K22 (helix h2) on one side, and by the side chains of N40 (C-terminus of helix h3) and K42 (loop h3–h4) on the other side (Fig. 3B). MuB NTD is rather acidic, with an isoelectric point (pI) value of 5.2. In the orientations shown in Fig. 3 the protein exhibits two acidic regions, a ‘‘top’’ patch provided by the carboxyl-terminus of residue A63 and by the side chains of residues E15, E17 and E18 (at loop h1–h2), and a smaller ‘‘bottom’’ acidic surface provided by residues D46 and E48 (N-end of helix h4) (Fig. 3A and B). The lateral surfaces of the domain have a positive electrostatic potential, particularly at both ends of helix h3 (Fig. 3B). 3.4. MuB NTD structure reveals a striking similarity to DNA-binding domains A search of the Protein Data Bank with the program DALI (Holm and Rosenstrom, 2010) showed that the overall fold of MuB NTD is highly similar to the structure of k repressor-like DNA binding domains (Interpro IPR010982) (Table 2). This protein superfamily groups a large number of prokaryotic and eukaryotic transcription factors with a similar a-helical domain and a HTH motif for DNA recognition. Within the k repressor-like family, and despite limited sequence homology (10–18%) (Fig. 4A), MuB NTD shows highest structural similarity with the POU-specific domain (POUS) of human octamer binding transcription factors (Oct) (Assa-Munt et al., 1993) (Table 2). Proteins of the POU family, which include human Pit-1 and Oct-1 and Caenorhabditis elegans Unc-86 as founding members, contain a bipartite DNA-binding POU domain formed by a POUS and a homeodomain (POUHD), tethered by a variable linker region (Fig. S1). MuB NTD superimposes with human Oct-1 POUS with an RMSD of 2 Å for 61 aligned Ca atoms (Fig. 4B). Both protein domains are monomeric and share a four a-helical fold, whereas k repressor has an additional fifth C-terminal helix responsible of protein dimerization (Fig. S1). In turn, POUS domains exhibit an unusual HTH motif with an extended loop and in this regard, the HTH motif of MuB is more similar to k repressor or to other members of the family, with a short 3-residue loop linking the two helices (Fig. 4C). Despite this difference in the HTH loop, highest sequence similarity between MuB and POUS was found at helix h3 (Fig. 4A). In Oct-1 POUS, this helix, predominantly its N-terminal half, enters in the major groove and interacts with the DNA through five highly conserved residues (Fig. S1) (Klemm et al., 1994), three of which contact the DNA bases and the other two interact with the phosphates (Figs. 4A and S1). Two additional residues outside helix h3 also interact with the DNA phosphates. Interestingly, MuB shows no sequence conservation with Oct-1 residues contacting directly with the bases, but T34 and S36 in helix h3, and S31 and R49 outside the helix, align structurally with POUS residues interacting non-specifically with the DNA (Fig. 4A).



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h4

T20 T19

N[ppm] 15

S36

115

120

0.8

Q55

G29

Q52 N44

N47

T12 Q23 N2 N40 E17 N16 N16 N40 S51 K22 E27 R56 S37 H62 I24 T53 K60 R7 V14 E59 L13 A8 V50 I35 Q52 L26/D41 L30 L10 W57 Y61 L54 R49 Q23 T34 Y43 D5 I6

S28 S31 S4 K42 E18 F21 N2 Q55 E15

R11

L58

I39

E48

M1

I3

F38

125

{1H}-15N NOE

110

R7ε

A25

0.6

0.4

0.2 A63

130 9

8

7 1

0

6

10

H[ppm]

20

30

40

50

60

Residue number

C C 60 N

C

20

h1 10

h2

h4

N

20

h1 60

10

h2

h4

40 1

40

30

h3 50

1

30

h3 50

Fig. 2. NTD structure determination by NMR. (A) 1H–15N HSQC NMR spectrum of uniformly 15N labeled MuB NTD in 20 mM sodium phosphate pH 7.5 and 0.1 M NaCl at 25 °C. Residue assignments are labeled according to the sequence numbering for the complete protein. Side chain assignments of asparagine and glutamine amides are connected with horizontal lines. (B) Heteronuclear {1H}–15N nuclear Overhauser effect (XNOE) of backbone amides. Secondary structure elements are indicated. (C) Stereoview of the superimposed 20 best structures of MuB NTD represented in Ca trace. The backbone is marked with spheres every ten residues and colored in rainbow gradient with the Nand C-termini colored in blue and red, respectively.

The structural similarity and sequence conservation with POUS strongly suggests a possible role of MuB NTD in DNA binding. To investigate whether the NTD could interact with the DNA we measured 1H–15N HSQC spectra of the NTD at 50 lM in the presence and absence of fivefold excess of a 14 bp double-stranded DNA containing the Oct-1 POU cognate binding sequence. Cross peaks from the NTD in presence and absence of DNA were indistinguishable, indicating no sign of binding (Fig. S2A). Additionally, no binding of the NTD to radiolabeled DNA was observed in an electrophoretic mobility shift assays (EMSA), even at high protein concentrations (1 mM) (Fig. S2A).

3.5. The NTD promotes MuB filament–filament interactions The poor solubility of MuB at low salt concentration and its tendency to precipitate upon addition of ATP are well known (Adzuma and Mizuuchi, 1991; Chaconas et al., 1985; Teplow et al., 1988). However, when working with the MuB construct lacking the NTD (MuB-DN; residues 65–312) (Mizuno et al., 2013), we noticed that this mutant does not precipitate upon binding of ATP. We followed up this observation in this work and found that the solubility of MuB wild-type (MuBwt) and MuB-DN in a buffer with low salt concentration (170 mM NaCl) is approximately similar, 0.2– 0.3 mg ml1, but upon addition of ATPS, more than 80% of MuBwt precipitates, whereas MuB-DN remains soluble (Fig. 5A).

We previously observed that shortly after addition of nucleotide (10 s), the filaments of MuBwt stick to each other in a parallel fashion, forming large bundles that were easily observed by negative staining EM (Dramicanin and Ramon-Maiques, 2013). The formation of these large filament aggregates was observed both in the presence and absence of DNA (Fig. 5B), and could explain the low solubility of the protein. MuB-DN, on the other hand, forms helical filaments of similar morphology to MuBwt (Mizuno et al., 2013) although polymerization appears to be somewhat slower (data not shown). Notably, these filaments remain independent and soluble even after overnight incubation with the nucleotide (Fig. 5C). The impairment of MuB-DN filaments to form bundles was confirmed at various salt (150–500 mM NaCl) and glycerol (5–20%) concentrations and at different pH values (pH 7–9), conditions in which MuBwt filaments aggregated readily (data not shown). These results clearly indicated that the NTD is involved in favoring filament–filament interactions. We have been unable to verify the direct interaction between the isolated NTD and MuB. The high salt concentration (>0.5 M) required for maintaining MuB in solution might have prevented the observation of the interaction by NMR (data not shown). On the other hand, the tendency of MuB-ATP filaments to precipitate has hindered the development of reliable assay to assess the interaction. To further test the participation of the NTD in the formation of filament bundles we introduced the mutation G33E into the



A

E17

E17

E18

7

E18

h1

E15

E15

C

K22

h2

N

L26

T32

90º

N40

h1

T32

h2

h3 S31

K22

L26

30 Å S36

G33

N40

G33 S31

K42

S37

h3

h4

K42

R49 E48

h4

D46

25 Å

B

E18

C-end

E48 D46

25 Å

E17

E18

E17

(A63) E15

K22

L26

L26

K22

T32 S36

90º S31

K42

N40

G33 S31

S37 K42

E48 D46

Fig. 3. MuB NTD structure. (A) Cartoon representation of MuB NTD with residue side-chains shown in sticks. Helices h1, h2, h3 and h4 are colored in blue, green, yellow and red, respectively. Residues forming the hydrophobic core are colored with the carbons in pink, whereas solvent exposed residues are colored with carbons in white. (B) Protein surface representation colored according to the electrostatic potential. Positive and negative surface potential are colored blue and red, respectively. The protein is oriented as in (A) and the approximate location of helix h3 is indicated with a dotted rectangle.

full-length MuB protein. This residue was selected based on an NTD–DNA model (see Section 4), where G33 would be the closest contact with the DNA. MuB-G33E mutant was expressed and purified as MuBwt (Fig. S3A). The solubility of the mutant is higher than MuBwt or MuB-DN and it is not significantly affected by the addition of ATPS or DNA (Fig. 5A). The mutation does not impair the ability of the protein to assemble into filaments, although MuB-G33E filaments do not associate into bundles, as observed for MuB-DN, (Fig. 5D). To test possible effects of the mutation in the stability or the correct folding of the NTD, we introduced the mutation in the MuB NTD construct (Fig. S3B). MuB NTD-G33E was produced in the same way as the non-mutated domain and we compared the 1 H NMR spectra of both proteins (Fig. S3C). The aliphatic and amide regions of the 1H NMR spectra are nearly identical, proving that the secondary and tertiary structures of both proteins are indistinguishable (Cavanagh, 2007). Therefore, we conclude that replacement of G33 by glutamate in helix h3 of the NTD directly prevents the interaction between MuB filaments.

4. Discussion The NMR structure reported here confirms the presence of a HTH motif within the N-terminal region of MuB (Miller et al., 1984). The HTH is one of the most frequent structural elements in DNA-binding proteins (Rohs et al., 2010), and consists of two a-helices joined by a short turn. The second helix is called ‘‘recognition helix’’ as it interacts specifically with the nucleotide bases through the major groove. Although the HTH fold is highly conserved, the structures embedding this motif vary among different protein families. The a-helical fold described here for MuB NTD was first observed in the N-terminal domain of bacteriophage k repressor (Pabo and Lewis, 1982), which became the paradigm of a large superfamily of DNA-binding domains. Within the k repressor-like family, MuB NTD shows closest similarity to POUS domains, particularly in those residues interacting non-specifically with the DNA. This similarity strongly suggested a DNA-binding function and led us to build a plausible model of the NTD–DNA interaction (Figs. 6 S1). We superimposed the NTD



8

Table 2 Search of MuB NTD homologs in the Protein Daba Bank with the program DALI. Protein name (residue range) POU transcription factors

PDB (chain)

Z-score

RMSD

Aligned residues

Identity (%)

Oct-1 (8–75) Oct-6 (252–319) Oct-4 (8–76) Pit-1a (8–74) Brn5 a (146–214) HNF-1-a a (105–171) HNF-1-b (111–177)

1E3O (C)

8.0

2.0

61

16

2XSD (C)

7.9

2.1

61

16

3L1P (A)

7.7

2.1

62

18

1AU7 (A)

7.7

2.1

60

17

3D1N (P)

7.3

2.2

62

16

1IC8 (A)

7.2

2.3

63

10

2H8R (B)

7.0

2.1

62

10

SATB1a (373–435) Phage 434 repressor (1–54) R-M controller Esp1396I (5–64) 434 Cro protein (1–54) Lac repressor (2–46) N25 Cro repressor (1–51) k repressor (12–71)

2O49 (A)

5.7

2.7

60

10

1R69 (A)

5.7

2.7

53

17

3G5G (K)

5.6

2.8

58

16

2CRO (A)

5.3

2.7

53

13

2PE5 (B)

3.7

2.4

45

16

2HIN (A)

3.6

2.7

49

8

1LMB (3)

3.6

3.2

54

15

a

a

a Oct, octamer-binding transcription factor; Pit, pituitary-specific positive transcription factor; Brn, brain-specific homeobox/POU domain protein; HNF, hepatocyte nuclear factor; SATB1, special AT-rich sequence binding protein 1; R–M, restriction–modification controller protein.

structure onto the backbone of Oct-1 POUS in complex with DNA (Chasman et al., 1999). The fitting was reasonable without further adjustments, except for a close contact of loop h3–h4. This loop is the most flexible region of the NTD (Fig. 2B and C), and could be easily accommodated by a small conformational change. In our model, the putative recognition helix of the NTD (helix h3) enters into the major groove of the DNA, and the small size of G33 – in the first helical turn of h3 – allows the close proximity of the two macromolecules. The side chains of lysines K22 and K42 embrace the double helix, interacting with the phosphates in different strands. Residues T20, S31, T34, S36, S37, N40, Y43, D46 and R49 (marked with red circles in Fig. 4A) are also predicted to contact the DNA phosphodiester backbone, in accordance with the limited sequence specificity of MuB (Greene and Mizuuchi, 2002a). Despite the attractive model, we failed to detect the putative interaction between the NTD and the DNA. However, this should not discredit the hypothesis that the NTD could be a DNA-binding domain. Indeed the POUS of Oct-1 and Pit-1 detached from their respective POUHD domains show almost undetectable affinity for the DNA (Ingraham et al., 1990; Verrijzer et al., 1992). It has been proposed that the linker to the DNA-bound POUHD maintains a high local concentration of free POUS domain, enhancing its binding to the DNA (Klemm et al., 1994). Since residues making specific contacts with the DNA are not conserved in MuB, we would expect the affinity of NTD for the DNA to be even lower than that of POUS. Perhaps, similarly to POU regions, tethering the NTD to the AAA+ helical filament would facilitate the binding of the NTD to nearby DNA. On the other hand, the negative charge of the top acidic patch near the putative DNA binding surface of the NTD (Fig. 6) could hamper the interaction due to electrostatic repulsion. This has been described for the CUTr1 domain of SATB1, another k repressor-like domain structurally similar to the NTD (Table 2). The isolated CUTr1 domain is very acidic and the interaction with DNA was only observed after introducing mutations that increased the pI value of the protein (Yamasaki

et al., 2007). Perhaps the affinity of the NTD for the DNA is enhanced in the context of full-length MuB, where the negative charge of the carboxyl-terminus would be absent and the acidic patch could be neutralized by a complementary positive surface in the AAA+ module. The location of the NTD within the MuB filament could not be determined by EM and it was proposed to be flexibly attached to the AAA+ module (Fig. 1A) (Mizuno et al., 2013). Only one EM reconstruction of MuB filaments without DNA showed a prominent density within the axial channel, which was tentatively assigned to the NTDs detached from the filament wall (Mizuno et al., 2013). As established here, the NTD is a compact domain, sufficiently small to fit in between the spirals of the AAA+ helix. Moreover, the 13 residue linker tethering the NTD to the AAA+ module is predicted to be disordered, and if extended, could expand the 50 Å distance from the outside of the filament to the inner channel. Thus, we do not foresee any steric impediment for the NTD to move into the AAA+ helix and reach the axial channel. The NTD has the HTH motif on the opposite side of the linker, a favorable location to contact the DNA within the filament, whereas the top acidic patch would be well placed to interact with the AAA+ surface facing the axial channel (Fig. 6A). These interactions could favor the assembly and stability of MuB filaments. Indeed, although filaments formed by MuBwt and MuB-DN look alike (Mizuno et al., 2013), we noticed that polymerization of the truncated mutant is slower than MuBwt, supporting the possible role of NTD in intra-filament interactions. Binding within its own filament does not explain how the NTD mediates the formation of filament bundles. We suspect that filament association is not favored through direct NTD–NTD contacts, since the isolated domain behaves as monomer in solution at high concentrations (Fig. 1C). Alternatively, we propose that the NTDs projecting from one filament might penetrate into an adjacent filament interacting with the AAA+ spirals and perhaps also with the DNA bound within the axial channel as explained above (Fig. 6B).



A

9

HTH motif

h1

h2

h3

h4

recognition helix

base-specific interactions

solvent exposed hydrophobic residue

backbone (non-specific) interactions

putative DNA interacting residues

hydrophobic core residues

Oct1-POUS

B

Oct1-POUS

MuB-NTD

MuB-NTD

C

C

C

C

h1

N

h2

h1

N

h4

h2

h4

h3

h3 N

N

λ repressor

C

λ repressor

MuB-NTD

MuB-NTD

C

C

h1

h2

h1

N

h2

N

h4

h4

h3

h5

h3

h5

N

N

Fig. 4. MuB NTD is structurally similar to k repressor-like DNA binding domains. (A) Sequence alignment of MuB NTD, Oct-1 POUS and k repressor N-terminal domain. The numbering of POUs sequence corresponds to that of the deposited structure. Residues are colored according to the helix they belong to, which are depicted as cylinders on top. The asterisks and dots below the sequences indicate that the MuB residue is conserved in two or only in one of the aligned proteins, respectively. (B and C) Stereodiagrams showing the backbone superposition of MuB NTD (in blue) to Oct-1 POUS (PDB 1CLT, in purple) (B), or to the k repressor N-terminal domain (PDB 1LMB, in green) (C).

The interaction of the NTD with the AAA+ helix must be sufficient for the association since bundles are also observed in the absence of DNA (Fig. 5B). Even if interactions were not strong, the high local concentration of NTDs (5.4 MuB subunits per helical turn) and the cooperativity of a zipper-like association would increase the avidity of filament cross-linking. These models are supported by results with mutant G33E, which behaves as if the NTD had been removed (Fig. 5). The replacement of G33 in the first turn of helix h3 by a glutamate does not interfere with MuB polymerization but inhibits filament clustering. Several phage repressors have a conserved glycine in the first turn of the recognition helix to allow a close association with

the DNA. If G33 fulfilled a similar role in MuB, as predicted by our NTD–DNA model, the insertion of a large and negatively charged residue in this position would cause steric hindrance with the DNA and an incorrect positioning of the NTD. The mutation also inhibits bundling of filaments formed without DNA, suggesting that perhaps the glutamate causes charge repulsion of the NTDs within the axial channel. Taken together, our results indicate that, far from being an apparently dispensable region, the NTD might have unpredicted roles in the higher-order assembly of MuB nucleoprotein filaments. In addition to facilitating protein polymerization through intra-filament interactions, the ability of the NTD in bridging



10

A soluble protein (mg·ml-1)

0.5

- ATPγS + ATPγS

0.4

+ DNA - ATPγS + DNA + ATPγS

0.3 0.2 0.1 0

MuB-wt

DNA (-)

MuB-G33E

DNA (+)

MuB wild-type

B

MuB- N

MuB- N

C

MuB-G33E

D

Fig. 5. NTD enables the formation of MuB filament bundles. (A) Protein solubility test. (B–D) Negative staining electron micrographs of filaments formed by MuBwt (B), MuBDN (C) and MuB-G33E (D) in the presence or absence of DNA. Only MuBwt filaments assemble into bundles. Scale bar, 100 nm.

MuB filaments expands the possibilities of the dynamic association of MuB with the DNA. It has been found that the DNA inside the filament cannot be used as a target DNA for transposition (Ge and Harshey, 2008; Mizuno et al., 2013) and a mechanism was proposed by which the coordinated ATP hydrolysis of several MuB subunits at the filament end could distort the DNA structure, favoring DNA bending and recognition by the transposome (Mizuno et al., 2013). The interaction between MuB filaments bound to nearby DNA sequences could further facilitate the bending of the intermediate sequence or the formation of a special DNA structure, thus assisting target DNA capture by MuA. Another possibility is

that the interacting filaments bridge distant DNA regions, causing the looping of long DNA sequences. It has been reported that effective interaction between MuA bound to the transposon ends and DNA-bound MuB requires the formation of DNA loops (Han and Mizuuchi, 2010). The relative stability of these loops was proposed to depend on direct interactions between MuA and MuB. However, we do not discard the possibility that interactions between MuB filaments or even interactions of the NTD with MuA or with MuA-bound DNA, could contribute to the stability of DNA looping. We also foresee the bundling of various filaments spread along a DNA molecule as an effective way of compacting the DNA. How



A

11

B E17 E18

E17

DNA

+

AAA

E15

AAA+

NTD K22

K22

L26

L26

G33

K42 G33

90º

R49

Fig. 6. Models of MuB–DNA interaction and filament bundling. (A) Binding of NTD to DNA based on the structure of Oct-1 POUS–DNA complex (PDB 1CQT) (see Supplementary Fig. S1 for details on the protein–DNA interactions). NTD is shown in surface representation colored according to the electrostatic surface potential and DNA is shown in sticks. (B) NTD-dependent clustering of MuB filaments. NTDs are represented as small black spheres hanging from the AAA+ helices. For simplicity, only two NTDs per helical turn are represented.

transposition and target immunity mechanisms could benefit from a close-packed DNA molecule is unclear. Future advances in understanding Mu transposition and immunity mechanisms will require functional analysis to investigate the importance of the NTD-mediated interactions between MuB filaments. 5. Accession numbers NMR conformation restraints and coordinates of the 20 best structures are deposited in the Protein Data Bank with accession number 2MQK. NMR assignment data is deposited in the BioMagResBank (BMRB) with accession number 25037. Funding This work was supported by the Spanish Ministry of Economy and Competitiveness (BFU2010-16812 and BFU2013-48365) and by the Spanish National Cancer Research Centre (CNIO) intramural program. MD has been a recipient of a predoctoral fellowship from ‘‘La Caixa’’ Foundation, BL-M is partially funded by Comunidad Autónoma de Madrid (S2010-BMD-2457, BIPEDD2) and SR-M is a researcher of the Ramón y Cajal program from the Spanish Ministry of Economy and Competitiveness. Acknowledgments We thank Dr. M.D. Moreno-Morcillo for critical reading of the manuscript; and Dr. A. Grande-García, A. Ruiz-Ramos and F. del Caño for help with site-directed mutagenesis, protein expression and for insightful discussion. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jsb.2015.07.004. References Adzuma, K., Mizuuchi, K., 1988. Target immunity of Mu transposition reflects a differential distribution of Mu B protein. Cell 53, 257–266. Adzuma, K., Mizuuchi, K., 1989. Interaction of proteins located at a distance along DNA: mechanism of target immunity in the Mu DNA strand-transfer reaction. Cell 57, 41–47. Adzuma, K., Mizuuchi, K., 1991. Steady-state kinetic analysis of ATP hydrolysis by the B protein of bacteriophage mu. Involvement of protein oligomerization in the ATPase cycle. J. Biol. Chem. 266, 6159–6167.

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MuB gives a new twist to target DNA selection.

MuB protein allosterically activates strand transfer by the transposase of phage Mu.

TICAM-1 has an N-terminal helical domain with structural similarity to IFIT proteins.

A MUB E2 structure reveals E1 selectivity between cognate ubiquitin E2s in eukaryotes.

Coverage of protein domain families with structural protein-protein interactions: current progress and future trends.

A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.

SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells.

Similarity between the DNA-binding domains of IHF protein and TFIID protein.

Structural and Biochemical Analysis of Protein-Protein Interactions Between the Acyl-Carrier Protein and Product Template Domain.

Extending Protein Domain Boundary Predictors to Detect Discontinuous Domains.

PTEN-PDZ domain interactions: binding of PTEN to PDZ domains of PTPN13.

The N Terminus of the Prion Protein Mediates Functional Interactions with the Neuronal Cell Adhesion Molecule (NCAM) Fibronectin Domain.

Inferring domain-domain interactions from protein-protein interactions with formal concept analysis.

Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions.

Domain atrophy creates rare cases of functional partial protein domains.

Structural and Biophysical Characterization of the Interactions between Calmodulin and the Pleckstrin Homology Domain of Akt.

Structural conservation of the PIN domain active site across all domains of life.

Structural domains of the extracellular domain of human nerve growth factor receptor detected by partial proteolysis.

Could structural similarity of specific domains between animal globins and plant antenna proteins provide hints important for the photoprotection mechanism?

Protein-protein interactions: structural motifs and molecular recognition.

The Intimin periplasmic domain mediates dimerisation and binding to peptidoglycan.

Structural and functional comparisons of retroviral envelope protein C-terminal domains: still much to learn.

Domain similarity based orthology detection.