R. sphaeroides are presented that enabled the phasing of a triple PAS domain construct. Together, these structures reveal the importance of α-helical PAS extensions for multi-PAS domain-mediated protein oligomerization and function.Crystal structures of two truncated variants of the transcription factor PpsR from  Rhodobacter sphaeroides is such a multi-PAS domain protein that, in addition to its three PAS domains, contains a glutamine-rich linker and a C-terminal helix–turn–helix DNA-binding motif. Here, crystal structures of two N-terminally and C-terminally truncated PpsR variants that comprise a single (PpsRQ-PAS1) and two (PpsRN-Q-PAS1) PAS domains, respectively, are presented and the multi-step strategy required for the phasing of a triple PAS domain construct (PpsRΔHTH) is illustrated. While parts of the biologically relevant dimerization interface can already be observed in the two shorter constructs, the PpsRΔHTH structure reveals how three PAS domains enable the formation of multiple oligomeric states , highlighting that not only the PAS cores but also their α-helical extensions are essential for protein oligomerization. The results demonstrate that the long helical glutamine-rich linker of PpsR results from a direct fusion of the N-cap of the PAS1 domain with the C-terminal extension of the N-domain that plays an important role in signal transduction.Per–ARNT–Sim (PAS) domains are essential modules of many multi-domain signalling proteins that mediate protein interaction and/or sense environmental stimuli. Frequently, multiple PAS domains are present within single polypeptide chains, where their interplay is required for protein function. Although many isolated PAS domain structures have been reported over the last decades, only a few structures of multi-PAS proteins are known. Therefore, the molecular mechanism of multi-PAS domain-mediated protein oligomerization and function is poorly understood. The transcription factor PpsR from  PAS domains are 100–120-amino-acid structural modules that occur in all kingdoms of life. Although PAS domains can exhibit low sequence identities among each other, they share a conserved three-dimensional fold consisting of a five-stranded antiparallel β-sheet surrounded by several short α-helices  present in the promoter region of PpsR-regulated genes  from R. sphaeroides. Based on secondary-structure predictions, we designed N- and C-terminally truncated PpsR variants that comprise a single, two and three PAS domains  and microscale thermophoresis (MST) measurements to determine and quantify the oligomerization states of the PpsR variants in solution. In conclusion, the PpsR structures reveal an intriguing PAS-mediated protein homo-oligomerization, demonstrating that the α-helical extensions significantly contribute to protein oligomerization in addition to the PAS cores. Additionally, the observed PpsRN-Q-PAS1 dimer organization appears to resemble a general architecture for the connection of PAS modules with a variety of effector or sensory domains. Furthermore, the long Q-linker helix, which is the result of a fusion of the N-cap of PAS1 with the C-terminal extension of the N-domain, features structural flexibility that plays an important role for signal transduction.Here, we report the determination of the three-dimensional structure of PpsR from 2.2.1.Escherichia coli  and was cloned into the pETM-11 expression vector using NcoI and NotI restriction sites. The resulting PpsR construct encodes the full-length protein including an N-terminal hexahistidine tag followed by a Tobacco etch virus (TEV) protease cleavage site (pET-His-TEV-PpsRfull). PpsRQ-PAS1, PpsRN-Q-PAS1 and PpsRΔHTH constructs were amplified by PCR using pET-His-TEV-PpsRfull as a template and the following primer pairs: 123_fw (5′-ATATATGGCCGAATTGCAGAAGTTCAGCAGCAGCTG-3′) and 257_rv (5′-ATATGCGGCCGCTTAATCTGCCGGATCAATCTGACACAGC-3′) for PpsRQ-PAS1, 1_fw (5′-ATATATGGCCGAATGGGTCTGGCAGGCGGTTC-3′) and 257_rv for PpsRN-Q-PAS1, and 1_fw and 379_rv (5′-ATATGCGGCCGCTTAACGGCTGGTATCACGAACAACC-3′) for PpsRΔHTH. The start and stop codons introduced by the forward and reverse primers, respectively, are underlined and the NcoI and NotI restriction sites are highlighted in bold. The truncated PpsR constructs were cloned into the pETM-11 vector as described for pET-His-TEV-PpsRfull, resulting in pET-His-TEV-PpsRQ-PAS1, pET-His-TEV-PpsRN-Q-PAS1 and pET-His-TEV-PpsRΔHTH, respectively.The gene encoding PpsR was synthesized with codon optimization for expression in 2.2.E. coli BL21 (DE3) cells (Invitrogen) were transformed with the different pET-His-TEV-PpsR plasmid DNAs. Single colonies grown on Luria–Bertani (LB) agar plates containing 50 µg ml−1 kanamycin were picked for inoculation of an overnight culture consisting of LB medium supplemented with 30 µg ml−1 kanamycin. The overnight culture was used to inoculate 12 l LB medium containing the same antibiotic. The cell cultures were grown to an optical density at 600 nm of 0.4 absorption units (AU) at 37°C, cooled to 18°C and the cells were further grown until the optical density reached 0.8 AU. Protein expression was induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. The cells were harvested by centrifugation 16 h post-induction.Chemically competent A  including cOmplete Protease-Inhibitor Cocktail (Roche). The cells were lysed using a microfluidiser (Microfluidics) and the lysates were clarified by ultracentrifugation at 185 500g at 4°C for 1 h.All cell pellets were resuspended in buffer full. The cleared supernatant was loaded onto an Ni2+–NTA Superflow (Qiagen) affinity column pre-equilibrated with buffer A. The resin was washed with ten column volumes (CVs) of buffer A and the bound proteins were eluted using 5 CVs of buffer B . PpsR-containing fractions were dialysed against 1 l buffer C  at 4°C overnight. In parallel, the hexahistidine tag was removed from the protein using TEV protease with a 1:30 molar ratio of TEV:protein, respectively. The cleaved tag and the histidine-tagged TEV protease were removed from the PpsR-containing solutions by chromatography on Ni2+–NTA resin. Subsequently, the flowthrough of PpsRfull only was additionally loaded onto a HiTrap Heparin column  equilibrated with buffer D . After washing the column with buffer D, the bound protein was eluted in a gradient to 75% buffer D supplemented with 850 mM NaCl over 10 CVs. Fractions containing PpsRfull and the Ni2+–NTA flowthrough of the truncated variants were concentrated using centrifugal filter units (Amicon) and were subjected to gel filtration on a Superdex 200 column  equilibrated with buffer D. The protein-containing fractions were collected, concentrated, aliquoted, frozen in liquid nitrogen and stored at −80°C.All hexahistidine-tagged PpsR variants were purified following the same protocol with an additional purification step for PpsRQ-PAS1 and PpsRΔHTH proteins were expressed in E. coli BL21 (DE3) according to the protocol of Van Duyne et al.  and reservoir solution consisting of 100 mM sodium cacodylate pH 6.5, 1.9%(w/v) polyethylene glycol (PEG) 3000, 200 mM MgCl2 after 1 d. However, individual crystals required up to 10 d to reach their maximal dimensions of ∼60 × 55 × 40 µm. Selenomethionine-substituted PpsRQ-PAS1 crystals appeared in a 2 µl 1:1 mixture of protein solution (3.5 mg ml−1) and reservoir solution consisting of 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 6.5, 3%(w/v) PEG 4000, 200 mM MgCl2, 5 mM DTE and reached their final size after 2 d. For cryoprotection and also for substitution of the cacodylate buffer, crystals obtained from both experiments were incubated in a solution consisting of 100 mM MES pH 6.5, 10%(w/v) PEG 3000, 200 mM MgCl2, 15%(v/v) ethylene glycol for 1 min and were subsequently flash-cooled in liquid nitrogen.Since previous attempts to crystallize PpsRN-Q-PAS1 crystals appeared from a 2 µl 1:1 mixture of protein solution (10 mg ml−1) and reservoir solution consisting of 1.6 M ammonium sulfate, 100 mM MES pH 6.5, 10 mMl-cysteine, 4%(v/v) 1,4-dioxane after 1 d. Individual plate-shaped crystals reached final dimensions of ∼150 × 30 × 5 µm after 5 d but grew in clusters. However, single plates could be isolated from these clusters and were briefly incubated in reservoir solution supplemented with 20%(v/v) ethylene glycol prior to flash-cooling in liquid nitrogen.PpsRΔHTH crystals with a hexagonal morphology started to grow from a mixture of 1.1 µl protein solution (30 mg ml−1) and 0.9 µl reservoir solution consisting of 1 M ammonium sulfate, 100 mM Tris–HCl pH 8.5, 6%(w/v) xylitol, 12%(w/v) glycerol, 1%(v/v) dioxane after 2–3 d. Individual crystals reached their final size of ∼400 × 100 × 80 µm within two weeks. Prior to flash-cooling in liquid nitrogen, native as well as selenomethionine-substituted PpsRΔHTH crystals were incubated for 5 min in buffer consisting of 50 mM sodium phosphate pH 6.8, 12%(w/v) glycerol, 3.0 M sodium malonate pH 7.Native as well as selenomethionine-substituted PpsR2.4.XDS program suite  experiments on crystals of selenomethionine-substituted protein using AutoSol in PHENIX  at 100 K. Indexing, integration and scaling of the data sets was performed using the N-Q-PAS1 was phased by molecular replacement (MR) using Phaser . The phases from MR were of sufficient quality to calculate an electron-density map based on diffraction data to 2.2 Å resolution that enabled building of the missing parts of the polypeptide chain. The structure was then further refined as described above for PpsRQ-PAS1 using three TLS groups for each protomer, corresponding to the N-domain (residues 6–120), the Q-linker (residues 121–157) and the PAS1 domain (residues 158–257).PpsRΔHTH was determined using a combined MR and Se-SAD approach. Statistics of the data collection, structure refinement and model quality of PpsRΔHTH and PpsRΔHTH Se-SAD have recently been published  in its dimeric form and the second encompassed a monomer of the C-terminal part (residues 145–257). Two molecules of the N-terminal dimer and four molecules of a monomeric C-terminal model were placed in the asymmetric unit, indicating a tetrameric PpsRΔHTH assembly. Although the electron-density map after MR was of insufficient quality for model building, the combination of MR phases (from AutoMR) with anomalous diffraction data  restraints were applied to the corresponding PpsR protomers A and D as well as B and C during initial rounds of model building and refinement, but were omitted during the end of the refinement to account for differences between the NCS-related protomers. TLS refinement was included , during the final refinement cycles.The structure of PpsRMolProbity , 4l9f (PpsRQ-PAS1 SeMet) and 4l9g (PpsRN-Q-PAS1). The PpsRΔHTH structure was previously deposited as PDB entry 4hh2  at the European Bioinformatics Institute .Statistics of the data collection, structure refinement and model quality of all structures are reported in Table 12.5.D, and 400 µl of a 20 µM protein solution containing the respective PpsR variants was loaded and fractionated at a flow rate of 0.5 ml min−1. Data analysis was performed using the ASTRA software provided by the manufacturer (Wyatt Technology).Individual PpsR variants were analysed using an HPLC (Waters) setup including a Superdex 200 10/300 GL column  connected to a MALS detector  combined with a refractive-index detector (Waters). The system was equilibrated with buffer 2.6.full and the truncated variants was quantified by microscale thermophoresis (MST) using a Monolith NT.115 (Nanotemper). The proteins were randomly labelled at their amine positions using NT-647 red fluorescent dye (Nanotemper), with approximately one label per monomer, as described in the Monolith NT protein labelling kit RED-NHS provided by the manufacturer (Nanotemper). 1:2 dilution series of unlabelled proteins were prepared over an appropriate concentration range using buffer D and were mixed with equivalent volumes of labelled proteins, resulting in final concentrations of 12.5 nM for PpsRfull and PpsRΔHTH as well as 10 nM for PpsRN-Q-PAS1 with respect to the labelled species. Standard treated capillaries (Nanotemper) were used for all experiments. Data for three individual experiments were averaged and evaluated using the quadratic equation of the law of mass action with the constraint of a fixed labelled species concentration.The oligomerization of PpsR3.3.1.ΔHTH  and the PAS1 domain  in the asymmetric unit. The 20 N-terminal residues are disordered and could not be modelled into the electron-density map. The remaining ordered part of the Q-linker region (residues 143–157) forms a helical element that corresponds to a classical PAS N-cap. Together with a molecule related by crystallographic dyad symmetry, PpsRQ-PAS1 forms a PAS dimer which features an assembly common to N-cap-comprising PAS domains .All initial attempts to solve the phase problem of PpsRN-Q-PAS1, amino acids 1–257; Fig. 1N-Q-PAS1 were obtained by MR using the PpsRQ-PAS1 dimer as a search model. Since the dimer arrangement of the PAS1 domain was virtually identical in both crystal forms, we obtained excellent phases and could model the remaining parts of the Q-linker and the N-domain into the electron-density map. The final model of PpsRN-Q-PAS1 contains a dimer in the asymmetric unit comprising residues 6–246 for protomer A and 8–250 for protomer B . Poor electron density was only observed in the β3N–β4N loop . Whereas unambiguous and interpretable electron density was observed for most regions of promoters A, B and C, weak electron density was observed for parts of the N-domain and PAS1 domain of protomer D. These parts were modelled taking into account information from the higher resolution structures and protomer A of the PpsRΔHTH structure that is related to protomer D by noncrystallographic symmetry. Ambiguous electron density was also observed in the loop connecting the PAS1 and PAS2 domains around residues 258–262 for protomers A, B and C, suggesting high flexibility of this region. However, this region could be modelled in protomer D, and superposition with the other protomers enabled the correct assignment of all PAS2 domains to the individual polypeptide chains. Finally, the five N-terminal residues as well as the C-terminal arginine of all protomers were disordered and could not be modelled into the electron-density map.The structure of the PpsRly Fig. 3d. Where3.2.Q-PAS1 structure revealed a typical PAS fold consisting of a five-stranded antiparallel β-sheet flanked by several helices. In addition to the PAS1 core, part of the Q-linker adopts an α-helical conformation corresponding to a PAS N-cap. This is important for dimer formation as observed for other N-cap-containing PAS domains  and supported the crystallographic dimer as biologically relevant. The main dimerization interface is formed by the N-cap, its connecting loop to the PAS core (residues 156–161) and β51 , as also reported for a subset of other PAS proteins .The PpsR72 Fig. 3b and suin Fig. 3a.Q-PAS1 dimer, we observed a dimeric arrangement of the two protomers in the PpsRN-Q-PAS1 structure . The PpsRN-Q-PAS1 dimer has a barbell-like quaternary structure with a protomer crossing along a central imperfect dyad axis. Owing to the protomer crossing, the N-domain and PAS1 domain of a single protomer are located on opposite sides of the dyad axis. The functional relevance of the observed dimer architecture is supported by a large number of contacts, which lead to a total BSA of 6180 Å2. The PpsRN-Q-PAS1 structure also allowed the identification of an additional PAS domain in the N-terminal part of PpsR, which was predicted based on sequence information from some PpsR homologues  as well as in β42 and the loop connecting the N-terminal capping helix of PAS2 (αP2) and β12 . In addition, the PAS2 N-cap also contributes to dimer formation by interacting with the corresponding N-cap of the second protomer. Notably, His275, which has been proposed to be involved in the coordination of a haem cofactor . This interaction results in the formation of a PpsR octamer  mediated by contacts of the N-domains, the Q-linkers and the PAS1 domains of the crystallographic and symmetry-related protomers A and B. The BSA of the octamer interface is 27 160 Å2, which is comparable to that of the tetramer interface (BSA of 26 870 Å2). Additional stabilization by the octamer interface is likely to be one reason for the better defined electron density observed for several regions of protomer A in comparison to protomer D.While the arrangement of the structural elements in the PpsRer Fig. 3e mediat1 of the PAS1 domain of protomer A (PpsRΔHTH), which is rotated by about 42° compared with all other PAS1 domains . This rotation is induced by contacts with a symmetry-related molecule in the octamer interface of the PpsRΔHTH structure. Comparisons of the N-domain, PAS1 and PAS2 domain dimers, including their N-caps, identified these structures as rigid dimer modules . Superpositions of all determined PpsR structures based on their PAS1 dimers revealed diverse orientations of the N-terminal helical part of the Q-linker, which results in different relative arrangements of the N-domain and PAS1 domain dimers in the determined PpsR structures . This is the result of inherent flexibility in a distinct hinge-like Q-linker region (residues 142–146), which is further reflected in subtle differences between the Q-linkers of the symmetrically arranged PpsRΔHTH protomers A and D as well as B and C . In contrast, the PAS1 cores and their corresponding N-caps are rigid modules that exhibit an identical conformation in all determined PpsR structures. Since α-helical extensions are not only present at the N-termini but also at the C-termini of some PAS domains  and the corresponding VicK S-helix PAS dimer can be superimposed with an average root-mean-square deviation (r.m.s.d.) of 4.2 Å for 2 × 131 Cα atoms, reflecting the similarities in dimeric arrangements . Furthermore, a comparable S-helix PAS organization can also be observed in a truncated structure of the HTR-like protein from Haloarcula marismortui , which resembles the PpsRQ-PAS1 structure. A search of the Pfam database . In both cases, this corresponds to approximately 1.8 times the molar mass of a monomer (42 kDa for PpsRΔHTH and 29 kDa for PpsRN-Q-PAS1). The eluting peaks did not show pronounced tailing and no continuous decrease in the molar mass signal was detected as observed for PpsRfull . Together, these findings indicate that both PpsR variants exist predominantly as dimers under the conditions of this experiment. However, the retention time of PpsRΔHTH was significantly reduced upon size-exclusion chromatography during protein purification at higher protein concentrations  and the elution profile showed peak tailing as observed for PpsRfull 2 ⇄ (PpsRΔHTH)4 based on the total dimer concentration. This value is in good agreement with the previously reported Kd value of 0.9 ± 0.3 µM determined for PpsRfull 2 transition with a Kd value of 0.25 ± 0.05 µM. In contrast, for PpsRQ-PAS1 we could not detect dimerization over the concentration range used in the MST experiments. Nevertheless, the concentrations used, for example, for crystallization also supported the formation of a biologically relevant PpsRQ-PAS1 dimer.To analyse and quantify the dimer–tetramer equilibrium of PpsRts Fig. 7. A Kd v4.R. sphaeroides, which reveals the importance of individual PAS domains and their α-helical extensions for PpsR oligomerization. Whereas the implications of the PpsRΔHTH structure in the context of the AppA–PpsR2 interaction and the regulation of gene expression in R. sphaeroides have recently been described . Such an octameric assembly has been reported to play a role in the binding of PpsR to its palindromic DNA target sequence  which are related by a crystallographic twofold axis, since the individual BSAs of the two tetramer assemblies are in comparable ranges. Nevertheless, the ABCD tetramer is most likely, since the truncated PpsRN-Q-PAS1 variant, which provides the entire interface for an A2B2 tetramer, is not able to form tetramers. In contrast, PpsRfull and PpsRΔHTH show the characteristic dimer–tetramer equilibrium, supporting a role of PAS2 in tetramer formation. Further support for the functional relevance of the ABCD tetramer is provided by interactions between the highly conserved helix α32 and the Q-linker  show that the deletion of seven N-cap residues disrupts the parallel dimer arrangement and results in a flipped, most likely nonphysiological, dimer . Blue-light illumination of VVD results in a series of structural changes that eventually affect the N-cap conformation and thus induce VVD dimerization  of E. coli  the PAS N-caps also help to stabilize the homodimer in its ligand-free and ligand-bound state, respectively.Like PpsR, many PAS proteins contain multiple PAS domains that either bind cofactors and sense environmental stimuli or exclusively serve as protein-interaction or signal transmission modules. Therefore, multi-PAS domain-mediated protein–protein interaction and oligomerization, as observed for PpsR, appears to be a common feature of many PAS-containing proteins. The PpsRet al., 2006et al., 2009bet al., 2006et al., 2006et al., 2009aet al., 2011et al., 2012et al., 20132 complex that results in structural changes of the N-terminal part of one Q-linker, whereas the conformation of the C-terminal part, which corresponds to one N-cap of the PAS1 dimer, and the N-domain dimer remain unaffected and resemble that observed in other PpsR structures . This suggests that the PAS domain dimers together with their N-caps function as rigid modules. The flexibility of a defined hinge region within the Q-linkers, however, supports the finding that the long Q-linker helices observed in the PpsRΔHTH structure result from a direct fusion of the C-terminal extensions of the N-domains with the N-cap of the PAS1 domains. Similar to the Q-linker of PpsR, SmVicK also possesses a long S-helix that originates from a fusion of a HAMP helix  with the N-cap of a PAS domain . Additionally, a similar S-helix PAS dimer arrangement is also observed for the HTR-like protein from H. marismortui . Although the N-terminal region of the S-helix and the adjacent PAS domain are not part of the HTR-like protein crystal structure, it is likely that the dimer arrangement of tandem PAS domains featuring characteristic N-caps is similar to that observed for PpsRN-Q-PAS1. Therefore, the PAS–S-helix–PAS dimer organization observed for PspR might not only be a conserved dimerization motif in a variety of multi-PAS containing signalling proteins, but might also be a general architecture for the connection of PAS domains to a variety of effector and sensory domains such as REC, GAF, HAMP, MEDS or PocR domains. For the engineered light-regulated sensor histidine kinase YF1, where an S-helix connects a LOV light-sensor to a dimerization/histidine phosphotransfer domain, it has been proposed that the sensed signal is transmitted along the coiled-coil S-helices in the form of torque or helical rotation  domain . Additionally, the PAS–S-helix–PAS dimer organization observed for PpsR appears to be a common architecture for the connection of PAS domains to a variety of effector and sensory domains in multi-domain signalling proteins. The signals sensed by such PAS domains are transmitted Q-PAS1, 4l9ePpsRPDB reference: Q-PAS1 SeMet, 4l9fPpsRPDB reference: N-Q-PAS1, 4l9gPpsRPDB reference: 10.1107/S1399004713033634/cb5045sup5.pdfSupplementary Figures S1-S3. DOI:


Anaeromyxobacter dehalogenans is a δ-proteobacterium found in diverse soils and sediments. It is of interest in bioremediation efforts due to its dechlorination and metal-reducing capabilities. To gain an understanding on A. dehalogenans' abilities to adapt to diverse environments we analyzed its signal transduction proteins. The A. dehalogenans genome codes for a large number of sensor histidine kinases (HK) and methyl-accepting chemotaxis proteins (MCP); among these 23 HK and 11 MCP proteins have a sensor domain in the periplasm. These proteins most likely contribute to adaptation to the organism's surroundings. We predicted their three-dimensional folds and determined the structures of two of the periplasmic sensor domains by X-ray diffraction. Most of the domains are predicted to have either PAS-like or helical bundle structures, with two predicted to have solute-binding protein fold, and another predicted to have a 6-phosphogluconolactonase like fold. Atomic structures of two sensor domains confirmed the respective fold predictions. The Adeh_2942 sensor (HK) was found to have a helical bundle structure, and the Adeh_3718 sensor (MCP) has a PAS-like structure. Interestingly, the Adeh_3718 sensor has an acetate moiety bound in a binding site typical for PAS-like domains. Future work is needed to determine whether Adeh_3718 is involved in acetate sensing by A. dehalogenans. Bacteria employ a variety of signal transduction proteins to sample their environment and initiate appropriate cellular responses. The most heavily utilized bacterial tools for signal transduction are the so-called two-component signal transduction systems , Fe(III), halogenated phenols etc.  from this organism. According to the Microbial Signal Transduction (MiST) database  -Gold (Stratagene), which harbors an extra plasmid, pMgk, encoding one rare tRNA corresponding to rare Arg codons, AGG and AGA. To produce the proteins, bacterial cultures were grown at 37°C, 190 rpm in 1 L of enriched M9 medium  and 25 mg each/L of the inhibitory amino acids l-valine, l-isoleucine, l-leucine, l-lysine, l-threonine, and l-phenylalanine  were added. Subsequently, protein expression was induced with 0.5 mmol/L isopropyl-β-D-thiogalactoside (IPTG), and the incubator temperature was increased to 18°C. Following overnight incubation, the cells were harvested by centrifugation and resuspended in lysis buffer  glycerol, 50 mmol/L HEPES pH 8.0, 20 mmol/L imidazole, and 10 mmol/L β-mercaptoethanol). Cells were disrupted by lysozyme treatment (1 mg/mL) and sonication, and the insoluble cellular material was removed by centrifugation. The SeMet-labeled protein was purified from other contaminating proteins using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography on an ÄKTAxpress system  as described previously  into 20 mmol/L HEPES pH 8.0 buffer, 250 mmol/L NaCl, and 2 mmol/L dithiothreitol (DTT).The periplasmic sensor domains of The purified and concentrated sensor domain proteins were used in identification of crystallization conditions. Sitting drop vapor diffusion method using drops composed of 0.4 μL protein plus 0.4 μL well solution were equilibrated against a reservoir of 140 μL of reagent solution. The crystallization plates were setup with the help of the Mosquito liquid dispenser . Preliminary conditions identified for Adeh_2942 and Adeh_3718 sensor domains with MCSG screens 1–4  were further optimized by inclusion of similar reagents from other commercial screens. The crystals used for data collection were grown by hanging drop vapor diffusion.Adeh_2942 sensor was crystallized from Wizard II reagent #11 . Crystal was cryoprotected by transfer to a solution containing 75 μL reservoir solution and 25 μL ethylene glycol for 15–30 sec prior to plunging in liquid nitrogen.Adeh_3718 sensor was crystallized from Membfac (Hampton Research) reagent #2  diluted by water as follows: 350 μL reagent solution + 150 μL deionized water. Cryoprotection was achieved by supplementing the reservoir solution with ethylene glycol as described above for the Adeh_2942 sensor.X-ray diffraction data were collected at the selenium edge at the Structural Biology Center 19-BM . Data were processed with the program HKL3000  proteins from A. dehalogenans sensor domains were predicted to be either helical, or Per-Arnt-Sim (PAS)-like or tandem PAS-like domains . Based on the expression levels and solubility tests (data not shown) under standard expression conditions, seven sensor domains, four from HK proteins  and three from MCP proteins  were selected for large-scale protein preparation. The sensor domains were produced in conditions that incorporate selenomethionine for structure determination by multiple wavelength anomalous dispersion (MAD) method. Crystallization trials were carried out for all eight purified sensor domains. Hit conditions were identified for three sensor domains . Optimizations of the hit conditions have led to data collection quality crystals for two  followed by their structure determination by X-ray crystallography. The structures of the two sensor domains are described below.Periplasmic sensor domain (residues 33–184) of chemotaxis protein Adeh_2942 was isolated, purified, and crystallized as described in the methods section. Adeh_2942 crystallized in space group C2 and diffracted X-rays to 2 Å resolution. Structure was determined by SAD method utilizing the selenomethionine residues and refined to an R-factor of 0.198 and R-free of 0.251. The crystallographic data and refinement statistics are presented in Table α atoms are overlapped. The four helices are formed by residues 38–75 (α1), 77–101 (α2), 104–134 (α3), and 137–179 (α4)  and the second one is the sensor domain of aspartate receptor, Tar  followed by the serine receptor Tsr sensor domain .DALI structural homology search  of chemotaxis protein, Adeh_3718 was isolated, purified, and crystallized as described in the methods section. Adeh_3718 crystallized in space group P31 (residues 41–68), α2 (residues 72–87), followed by two small β-strands β1 (residues 96–100) and β2 (103–106), helix α3 (residues 110–114), helix α4 (residues 128–141), β3 (residues 142–151), β4 (residues 156–167), and β5 (residues 171–180). The structure of Adeh_3718 sensor has revealed a fully occupied acetate ion moiety bound to the protein in this binding site  and a sensor domain of chemoreceptor TlpB from Helicobacter pylori involved in pH sensing .Structural homology search with the program DALI has returned a number of PAS-like sensor domain structures from PDB. The top two closest hits are a sensor domain of an MCP protein of unknown function from A. dehalogenans strain 2CP-C. The sensor domains are part of signal transduction systems of HK and MCP proteins; they are located in the periplasm flanked by two transmembrane segments as shown in the schematic  Alltic Fig. , (2) PAStic Fig.  and hemeA. dehalogenans are shown in a pie chart representation  were crystallized and their structures determined by X-ray diffraction. In both cases, the structures confirmed the predictions from the corresponding sequences.No significant matches were found in the Pfam database for the sensor domain of Adeh_2942, however, FFAS03 server predicted it to be helical. Indeed, the crystal structure of Adeh_2942 revealed a helical bundle. In general, the sensor domains of signal transduction proteins have either weak or no oligomeric association in solution. In case of Adeh_2942 sensor, the crystallization process selected a head-to-tail dimer; in contrast the well-studied sensor domains of chemotaxis proteins, aspartate receptor, Tar , 4exo, the authors have modeled a pyruvate molecule in a similar binding site. In addition, in case of tandem PAS-like domains, the membrane-distal PAS-like domain also shown to bind small molecules in similar binding sites . Although acetate is a frequently used electron donor for organisms such as A. dehalogenans, a chemoreceptor for acetate has not been reported. He and Sanford  and helical bundle sensor domain tertiary folds. The main question then is whether the signal transduction mechanism used is specific to a given fold of the periplasmic signal detection domain. If so, is there one activation mechanism for each of the different sensor domain folds? Another pertinent question is how many of the signal transduction proteins directly sense a stimulus and how many require interaction with another protein  was found to have a helical bundle structure, and the other Adeh_3718 sensor (MCP) has a PAS-like structure. Interestingly, the Adeh_3718 sensor has an acetate moiety bound in the typical binding site of PAS-like domains. The possible implications of acetate sensing by this protein await experimental verification.In summary, we have predicted the structural folds of periplasmic sensor domains of 24 HK and 12 MCP proteins of


RLIP76 is an effector for Ral small GTPases, which in turn lie downstream of the master regulator Ras. Evidence is growing that Ral and RLIP76 play a role in tumorigenesis, invasion, and metastasis. RLIP76 contains both a RhoGAP domain and a Ral binding domain (GBD) and is, therefore, a node between Ras and Rho family signaling. The structure of the RhoGAP-GBD dyad reveals that the RLIP76 RhoGAP domain adopts a canonical RhoGAP domain structure and that the linker between the two RLIP76 domains is structured, fixing the orientation of the two domains and allowing RLIP76 to interact with Rho-family GTPases and Ral simultaneously. However, the juxtaposed domains do not influence each other functionally, suggesting that the RLIP76-Ral interaction controls cellular localization and that the fixed orientation of the two domains orientates the RhoGAP domain with respect to the membrane, allowing it to be perfectly poised to engage its target G proteins.  •The structure of the RLIP76 RhoGAP-Ral binding domain dyad has been solved•The interdomain linker contacts both domains and fixes their orientation•The RhoGAP domain is a poor GAP for Cdc42 and Rac1 in vitro•Ral and Rho family proteins can interact simultaneously with RLIP76 RLIP76 is an effector for Ral small GTPases that contains a RhoGAP domain adjacent to a Ral-binding domain. Rajasekar et al. solve a structure and reveal that the linker between the two domains is structured, fixing their orientation. This orientation allows them to engage their target G proteins. The Ral interacting protein (RLIP76)/Ral binding protein  is a downstream effector of the Ral GTPases, which themselves lie downstream of the key regulator small G protein Ras. Activated Ras has the ability to initiate a cascade of signaling pathways due to its capacity to interact with several different groups of effector proteins, the best studied of which are the Rafs, PI3 kinases, and RalGEFs. Despite the supreme ability of Ras to transform cells, of all the Ras effector families, only the RalGEFs can transform immortalized primary human fibroblasts, and this activity requires Ral . RalGEF RLIP76 is a multidomain, multifunctional protein that was first identified for its ability to interact with activated Ral GTPases . The RalRLIP76 is usually located in the cytoplasm, but translocates to the membrane upon activation by Ral proteins . It contWhether RLIP76 functions as a modular protein with independent domains, so that each domain is free to interact with its own interacting partner(s), or whether the domains interact among themselves and regulate each other is currently unknown. As the RhoGAP domain interacts with Rho-family small G proteins and the GBD interacts with the Ral proteins, RLIP76 could act as an intersection point for these distinct signaling pathways, and juxtaposition of the GAP and GBD domains could be the strategy employed by RLIP76 to coordinate regulation of both pathways.We have solved the structure of the RLIP76 GAP-GBD “didomain” with the aim of understanding any communication between the juxtaposed domains. The GBD in the dyad retains the same structure as the free GBD, and the RhoGAP domain adopts the canonical RhoGAP conformation, allowing the identification of the crucial catalytic residues. However, as RLIP76 has very poor GAP activity, we have also investigated the catalytic activity of the GAP domain and GAP-GBD didomain in vitro. Unexpectedly, the structure shows that the linker between the two domains is well defined and in a fixed conformation, sandwiching the two domains together in a locked configuration with respect to each other. Despite this, we find no evidence that either domain influences the functional activity of the other, and suggest rather that Ral binding to the RLIP76 GBD serves to localize RLIP76 to the correct cellular membrane and simultaneously fixes the orientation of the RLIP76 RhoGAP domain relative to that membrane, such that it is perfectly positioned to engage its Rho-family substrate, which will be tethered at the same location.The resonances of the GAP-GBD didomain (residues 184–446) were assigned as reported elsewhere . The finThe GAP domain (residues 184–368) is formed by nine α helices arranged in a typical RhoGAP fold , usually2KWI) is ∼1.0 Å. Thus, the overall architecture of the G protein binding domain is preserved in the longer construct. This is in agreement with our observations that the GAP-GBD didomain and the GBD single domain bind to RalB with the same affinity of ∼200 nM (The GBD (residues 393–446) in the didomain is a coiled coil, which is similar to the structure that we previously observed both in isolation and in complex with RalB . The bacThe orientation of the two single domains that make up the GAP-GBD didomain is well defined in the family of structures due to a number of distance restraints observed between each domain and the linker that connects them. The final α helix of the canonical RhoGAP domain finishes at Phe368, and the next nine residues (Gly369–Met377) contact the surface of the GAP domain comprising helices αE and αA and the linker between αA0 and αA C. SeveraThere are few distance restraints between the two domains because they do not form a large buried surface area at their interface, and indeed only come together where Met377 in the linker acts as a hasp between the GAP and the N-terminal α helix of the GBD. Met377 contacts Tyr204, which is sandwiched between Phe407 and Leu408, and distance restraints between Met377 and Phe407 reinforce the contacts C. Long-r−1, respectively, similar to values previously obtained by real-time experiments , 9-fold (L67A), and >20-fold (Y64A). Mutations of Val36Cdc42 and Leu70Cdc42, which are at the periphery of the loop interface, do not affect the binding affinity, indicating that although they make contacts with the GAP domain, they are not thermodynamically essential. These data highlight the importance of the interactions between the switch regions of Cdc42 and the αF-αG loop of p50 RhoGAP for successful formation of the complex. None of these contacts can be formed by RLIP76. We therefore reasoned that the shortened loop in RLIP76 might lead to an inability of the GAP domain to engage Cdc42 and Rac1, whereas p50 RhoGAP binds to Cdc42 and Rac1 with affinities of 24 and 18 nM, respectively , which would be expected for a full competitor. The differences observable between the partial competitors RLIP76 GAP and RLIP76 didomain and a pure competitor, such as free p50 RhoGAP itself, are apparent , and the affinity between Rac1 and the RLIP76 GAP insert was essentially the same (We endeavored to determine whether reinstating a longer loop between helices αF and αG in RLIP76 could restore Cdc42 binding and thus improve RLIP76 GAP activity. Residues Gln351–Ser353 (Q-I-S) of the RLIP76 GAP domain were replaced by the equivalent sequence Leu401–Ile416 in p50 RhoGAP (L-W-A-K-D-A-A-I-T-L-K-A-I-N-P-I). The refolded protein was assessed by circular dichroism and shown to be helical , but it had no effect on the Cdc42 GAP activity. When higher concentrations of the RLIP76 GAP insert domain were used, close to stoichiometric levels compared to the Cdc42 concentration, the RLIP76 GAP insert actually inhibited the GTPase when compared to its intrinsic rate of hydrolysis D. The inTaken together, these data demonstrate that engineering the αF-αG loop from p50 RhoGAP into the RLIP76 GAP domain is sufficient to impart a measurable increase in binding affinity of RLIP76 GAP for Cdc42. Nevertheless, even this engineered form of RLIP76 GAP, which binds more tightly to Cdc42, cannot behave as an efficient GAP. Furthermore, the stabilization of the Cdc42 switch regions by the αF-αG loop actually inhibits the GTPase activity when it is present in RLIP76 GAP, presumably because it binds Cdc42·GTP to form an unproductive complex.1AM4) to dock Cdc42 to the RLIP76 GAP domain. The GBD in this model was then superimposed onto the GBD in our structure of the RalB-GBD complex (PDB ID code 2KWI). The resulting complex model  but reproducible decrease in activity in comparison to the GAP domain alone. Although it does not seem likely that such a small decrease would be functionally significant in vivo, it does show that the RLIP76 GAP domain has the capacity for slightly higher activity in the absence of the GBD. The GBD makes no contacts with the catalytic residues in the GAP domain, so we assume that this influence is a long-range, allosteric effect that prevents the subtle rearrangements important for formation of the transition state of the reaction. We also examined the effect of the GAP domain on the ability of the GBD to bind its partner, RalB. The binding affinities of the GBD and the didomain for RalB are identical, so the ability of the GBD to bind Ral proteins is unaffected by the presence of the GAP domain.As we expected any influence of each domain on the other to be regulatable, we investigated whether the activity was altered as a consequence of the presence of binding partners. The GAP activity of the didomain toward Cdc42 and Rac1 remains unaltered in the presence of RalB; that is, binding of RalB to the GBD does not alleviate the small negative influence of the GBD on the GAP activity. We cannot exclude the possibility, however, that the presence of Ral proteins might regulate the activity of the GAP domain toward other, as yet unidentified, Rho-family proteins.Thus, it would seem that although the two domains of RLIP76 are both juxtaposed and tethered in a fixed conformation, neither seems to have a large influence over the known functions of the other. We therefore conclude that the function of the GBD is to bind to one of the two Ral isoforms, which then acts to localize RLIP76 to a particular cellular compartment. At its particular location it then encounters the relevant substrate for the GAP domain. Our model of the tripartite complex reveals that the orientations of the domains would facilitate membrane localization by Ral with synchronous binding of Rho-family substrates by RLIP76.Human RLIP76 GAP (residues 184–380) and GAP-GBD domains (residues 184–446) were expressed and purified as described previously .351QIS353 of RLIP76 were replaced with 401LWAKDAAITLKAINPI417 from p50 RhoGAP. Mutagenesis was performed using a QuikChange Lightning mutagenesis kit (Agilent), following the manufacturer’s instructions. The RLIP76 GAP domain with the inserted F-G loop from p50 RhoGAP was expressed in Escherichia coli BL21 (DE3) in inclusion bodies, which were washed in 2% Triton and 2 M NaCl and solubilized by resuspension in 6 M GuHCl (pH 7.5). The protein was refolded by rapid, 50× dilution at 4°C into 1.5 M 1-(3-sulfopropyl)-pyridinium betaine (Raschig-GmbH), stirred at 4°C overnight, concentrated, and further purified on a 16/60 S75 column.The F-G loop from p50 RhoGAP was inserted into the pET16b RLIP GAP domain construct using site-directed mutagenesis. Residues Mutations of cysteine residues in the GAP-GBD didomain were carried out using the QuikChange Lightning Multi Site-Directed mutagenesis kit (Agilent) following the manufacturer’s instructions. Cys216 and Cys227 were mutated to alanine; Cys291 and Cys313 were mutated to serine.All other expression constructs have been described elsewhere .All NMR experiments were collected and analyzed as described . Backbon15N-HSQC experiments or by using the ARTSY method  were measured on a Bruker DRX600 by partially aligning the protein in 13 mg/ml Pf1 phage (Asla Biotech). RDCs were measured by comparing peak positions in TROSY and decoupled Y method . InitialY method . The val15N-labeled protein with 20 mM MTSSL at 4°C overnight in 20 mM Tris-HCl (pH 7.9) and 100 mM NaCl. MTSSL loading was confirmed by mass spectrometry. 15N-TROSY HSQC spectra were recorded on unlabeled and MTSSL-labeled protein. Cross-peak intensities were measured, and the ratios were used to generate distance restraints  methanethiosulfonate; Enzo LifeSciences) was attached to Cys411 by incubating the straints  between 2 was added to 100 mM. The reaction mixture was passed through a G25 spin column to remove excess GTP and used immediately in the assays. The release of phosphate from Rac1 or Cdc42 was monitored at 360 nm using the EnzChek phosphate assay kit (Invitrogen) to quantify inorganic phosphate released in the reaction. The GAP activities of the RLIP76 didomain, RLIP76 GAP domain, and the GAP domain from p50-RhoGAP toward wild-type Rac1 (25 μM) and wild-type Cdc42 (25 μM) were monitored simultaneously on a microplate spectrophotometer . All experiments were performed at 25°C in 50 mM Tris-HCl (pH 7.5), 10 mM NaCl, and 1 mM MgCl2.Rac1 and Cdc42 were GTP loaded by incubation in a 10× molar excess of GTP, 60 mM ammonium sulfate, 20 mM EDTA, 20 mM Tris-HCl (pH 7.4), and 50 mM NaCl for 1 min at 37°C. Then the mixture was briefly incubated on ice and MgCl3H]GTP. Binding of the G protein to the His-RLIP76 constructs brings the labeled nucleotide close enough to the scintillant to obtain a signal. Apparent Kds for Q61L Rac1/Cdc42·[3H]GTP were measured as described previously  in the presence of Q61L Rac1/Cdc42·[eviously  by varyid errors . The inteviously .3H]GTP⋅Rac1 or [3H]GTP⋅Cdc42 and 30 nM GST-p50 RhoGAP immobilized on fluoromicrospheres via an anti-GST antibody (Invitrogen). The added RLIP76 or free p50 RhoGAP proteins compete with the GST-p50 RhoGAP-[3H]GTP⋅Rac1/Cdc42 interaction, abolishing the scintillation signal. The highest concentrations of competitor used were RhoGAP, 16 μM; His-RLIP76 GAP, 31.25 μM; His-RLIP76 didomain, 27.3 μM; and His-RLIP76 GAP insert, 17.5 μM. In each case, a blank was performed in the absence of GST-p50 RhoGAP GBD. For affinity determination, data points were obtained for at least ten different competitor concentrations. The Kd value and its standard errors were obtained by fitting the dose-response curve to binding isotherms that describe competition between two proteins binding to one site on another protein and account for mutual depletion of the interacting components. The Kd values for the GST-p50 RhoGAP-Rac1/Cdc42 interactions were obtained from direct-binding SPAs (For competition assays, His-RLIP76 GAP, His-RLIP76 GAP-GBD didomain, His-RLIP76 GAP insert, or free RhoGAP was titrated into a mixture of 30 nM [ing SPAs . The equing SPAs  and haveing SPAs .D.O. and H.R.M. conceived the project. L.J.C. made constructs and performed SPAs. K.V.R. made NMR samples and performed GAP assays. D.N. ran the NMR experiments. K.V.R. and H.R.M. analyzed NMR data and solved the structure. D.O. and H.R.M. characterized the GAP insert mutant. K.V.R., D.O., and H.R.M. wrote the manuscript.


Nef exists as small oligomers near membranes and associates into higher oligomers such as tetramers or hexadecamers in the cytoplasm. Earlier structures of the Nefcore in apo and complexed forms with the Fyn-kinase SH3 domain revealed dimeric association details and the role of the conserved PXXP recognition motif (residues 72–78) of Nef in SH3-domain interactions. The crystal structure of the tetrameric Nef reported here corresponds to the elusive cytoplasmic stage. Comparative analyses show that subunits of Nefcore dimers (open conformation) swing out with a relative displacement of ∼22 Å and rotation of ∼174° to form the ‘closed’ tetrameric structure. The changes to the association are around Asp125, a conserved residue important for viral replication and the important XR motif (residues 107–108). The tetramer associates through C4 symmetry instead of the 222 symmetry expected when two dimers associate together. This novel dimer-tetramer transition agrees with earlier solution studies including small angle X-ray scattering, analytical ultracentrifugation, dynamic laser light scattering and our glutaraldehyde cross-linking experiments. Comparisons with the Nefcore—Fyn-SH3 domain complexes reveal that the PXXP motif that interacts with the SH3-domain in the dimeric form is sterically occluded in the tetramer. However the 151–180 loop that is distal to the PXXP motif and contains several protein interaction motifs remains accessible. The results suggest how changes to the oligomeric state of Nef can help it distinguish between protein partners.HIV-1 Nef modulates disease progression through interactions with over 30 host proteins. Individual chains fold into membrane-interacting N-terminal and C-terminal core (Nef The 30-residue loop mentioned above is disordered in the complexes. This loop contains distinct motifs such as the di-Leu and EE motifs as also acidic residue clusters that interact with different sets of host/accessory proteins Each Nef chain folds into two domains closed’ oligomeric form has not been reported so far. This has led to a gap in understanding, in molecular terms, as to how Nef modulates its interactions with partner proteins. We report the crystallization of the full-length HIV-1 Nef after experimentation with naturally occurring Nef variants from patient samples and were successful with one such sample. This Nef variant was earlier shown by our groups to exhibit pathogenicity in a C. elegans model closed tetrameric association in the structure. The transition from a dimer to a tetramer involves large changes to the dimeric association to form a tetramer with 4-fold symmetry. The molecular details of the dimer-tetramer transitions help explain how the protein can distinguish between different sets of protein partners when it is in the membrane-associated and cytoplasmic stages respectively. To our knowledge this is the first example where a functional dimer-tetramer transition deviates from the expected 222 symmetry to 4-fold symmetry. These large changes to the intersubunit spatial disposition involve Asp125, a highly conserved residue known to be important for viral replication and the conserved XR motif (where X is Lys or Arg) core—FynSH3 complexes Despite earlier efforts, structural elucidation of a full-length Nef as also of the elusive ‘GenBank with accession number: GQ184340. This latter Nef variant (RP14) was also shown to be pathogenic in a C. elegans model We attempted crystallization of the full-length HIV-1 Nef (RP14) with several cloned samples derived from patients and were successful with the sample with sequence as deposited in the in vivo studies r.m.s.d values ranging between 0.69 and 0.83 Å between themselves and earlier reported structures of the domain. These values are not indicative of substantial changes to the tertiary structure as they are in the range of the average co-ordinate errors of the respective compared structures. On the other hand, the N-terminal anchor domain is disordered in the full-length structure. The presence of the full-length protein in the crystals was however confirmed by crushing a couple of crystals after washing them and running an SDS PAGE gel  now interacts with the second R residue of the XR motif  with two interacting peptides derived from the signalling ζ subunit of the T-cell receptor (TCRζ) It is perplexing as to how Nef mediates the interactions with a large number of proteins, especially given its rather small size and how it results in diverse functional consequences. It is generally agreed that conformational changes based on the cellular location of Nef plays an important role. Interaction motifs are distributed in the protein in primarily two regions; the first involves the PXXP motif and the other involves motifs situated in the 30-residue loop. The oligomeric changes exhibited by Nef were associated with the enforcing of the multiple distinct functions of the protein closed’ about the structure of the cytoplasmic form of Nef and that the protein controls interactions with different partners in novel ways. The shielding of the PXXP motif that interacts with the SH3 domains in the tetramer is unanticipated. We know of no other example where a dimer breaks its 2-fold symmetry to form a 4-fold symmetric tetramer. The unprecedented changes however retain the key interactions of Asp125 with the XR motif that were earlier shown to be essential for viral replication Overall, the present work shows that there is nothing ‘The study was approved by the Human Ethical Committee of the ‘Chhatrapati Shahuji Maharaj Medical University , Lucknow-226003 (UP) India, with Ref. Code: XVIII ECM/P9. The written informed consent was obtained from all study participants.5′-CCCTAYAACCMCARAGYCARGG-3′ and outer reverse LTRR 5′-GACTACGGCCGTCTGAGGGATCTCTAGYTACCA-3′ and Inner forward Primer NOFP 5′-ATACCTASAMGAATMAGACACARGG-3′ and inner reverse primer NORP 5′-CTGCTTATATGCAGCATCTGAGGG-3′) Peripheral Blood Mononuclear Cells (PBMCs) were isolated from a random HIV-1 infected individual, after informed consent, using the standard Ficoll density gradient centrifugation method (Sigma). The study was approved by the Human Ethical Committee of the ‘Chhatrapati Shahuji Maharaj Medical University’ , Lucknow-226003 (UP) India, with Ref. Code: XVIII ECM/P9. The written informed consent was obtained from all study participants. Genomic DNA was isolated from fresh PBMCs by a genomic extraction kit (Invitrogen). Nested PCR approach 5′-CATGGATCCCAGCAGCAGAAAGGAGA-3′ and reverse primer NX1R CCAGGTCTAGACCCAGCGGAAAGTCCC-3′ with BamHI restriction sites at their overhangs and subsequently subcloned into the pET28a bacterial expression vector (Novagen). The pET-28a-Nef plasmid containing hexahistidine tags was transformed into the C41 bacterial strain. The single colony culture was induced by the addition of 0.8 mM IPTG at 37°C for 6 hrs. Before purification the expression and solubility was checked on SDS-PAGE. The protein was purified by affinity chromatography using a Ni-NTA column and Tris-Cl (25 mM pH 8.8), NaCl (50 mM) buffer. The protein eluted at 350 mM imidazole concentration and was subjected to 12% SDS PAGE analysis where the Page Ruler™ Prestained Protein Ladder (Fermentas Cat. SM0671) was used as a molecular weight marker (The Nef gene was amplified and cloned into the TA cloning vector (Invitrogen) using forward primer P14BF t marker . SubsequThe purified Nef protein was incubated with increasing concentrations of Glutaraldehyde  on ice for 15 minutes. Respective samples were prepared and run on 12% SDS-PAGE. The gel was visualized after silver staining it. The gel was subjected to western blotting using anti-His antibody to unambiguously identify the oligomeric bands .322  and diffract to 3.5 Å. The data collection and refinement statistics are summarized in 3/Da or solvent content of about 48.7% suggested that 2 molecules were there in the asymmetric unit.Crystallization trials using the hanging drop method (2∶1 ratio of protein and precipitant solutions) were carried out using the purified Nef at a concentration of ∼3.7 mg/ml. Various constructs based on the Nef variants from patient samples were used for crystallization trials and ultimately we were successful with one such construct for reasons outlined in ‘http://www.bioinf.org.uk/software/profit) and parts of 1AVV, which structurally align well, were identified visually. Non-conserved residues were converted to alanine in the search model. Data in the resolution range 29.6-3.5 Å were used for molecular replacement calculation. Calculations were carried out in all the sub-groups of P622. The correct solution was in the space group P6322 and the packing was examined carefully. There are two independent chains in the asymmetric unit. There was also enough space to accommodate the N-terminal domain that was yet to be placed. Calculations involving the available myristoylated and non-myristoylated structures of the N-terminal domain were not successful despite various parameters like the resolution range etc being changed. An examination of the electron density maps at this stage suggested that the N-terminal domains might be disordered as suggested by weak trailing density at the start of the C-terminal core domain. The core domain itself was well defined in the electron density maps. We therefore started model building with a view that improved phases after model building might result in better maps for modeling the N-terminal domain. Ultimately we realized that the N-terminal domain is disordered, although it is present in the crystals (The structure was solved using molecular replacement (MR) techniques employing the program Phaser crystals . A long http://www.rcsb.org) with accession codes 2XI1 and R2XI1SF respectively.The coordinates and structure factors have been deposited in the Protein Data Bank The Nef protein was crosslinked by glutaraldehyde with 0.0% (control), 0.025%, 0.05%, 0.075%, 0.1% concentrations respectively and subsequently run on 12% SDS-PAGE. The gel was silver stained to visualize the different oligomeric forms of Nef protein. (B) Since the purified protein contains a hexa-His tag, we unambiguously identified the oligomeric forms by western blotting with anti-His antibody.(PDF)Click here for additional data file.Figure S2SDS-PAGE gel of crushed HIV-1 Nef crystals (right) run along with the molecular weight marker from Fermentas. A sample gel from a purification run is shown on the left for comparison. Lane L5 corresponds to the purified eluted protein from the Ni-NTA column while lanes L1-L4 correspond to the other stages of purification including the wash cycles.(PDF)Click here for additional data file.


In vitro Cdk2 inhibition assays showed that the p27 variants exhibited disparate inhibitory potencies. Furthermore, when over-expressed in NIH 3T3 mouse fibroblast cells, the less helical p27 variants were less effective in causing cell cycle arrest relative to the wild-type p27. Our results indicate that the nascent helicity of sub-domain LH plays a key role mediating the biological function of p27.p27 regulates the activity of Cdk complexes which are the principal governors of phase transitions during cell division. Members of the p27 family of proteins, which also includes p21 and p57, are called the Cip/Kip cyclin-dependent kinase regulators (CKRs). Interestingly, the Cip/Kip CKRs play critical roles in cell cycle regulation by being intrinsically unstructured, a characteristic contrary to the classical structure-function paradigm. They exhibit nascent helicity which has been localized to a segment referred to as sub-domain LH. The nascent helicity of this sub-domain is conserved and we hypothesize that it is an important determinant of their functional properties. To test this hypothesis, we successfully designed and prepared p27 variants in which domain LH was either more or less helical with respect to the wild-type protein. Thermal denaturation experiments showed that the ternary complexes of the p27 variants bound to Cdk2/Cyclin A were less stable compared to the wild-type complex. Isothermal titration calorimetry experiments showed a decrease in the enthalpy of binding for all the mutants with respect to p27. The free energies of binding varied within a much narrower range.  Ink4b (p15), Ink4a (p16), Ink4c (p18), Ink4d (p19) Waf1/Cip1/Sdi1/Cap20 (p21), Kip1 (p27), Kip2 (p57) 1 to S transition Strict temporal control of cell cycle phase transitions is governed primarily by a family of highly conserved serine/threonine kinases termed the Cyclin-dependent kinases (Cdks) 10 helix on binding Cdk2. However, it is important to note that even though they are largely disordered prior to binding their targets, the Cip/Kip proteins have been shown in isolation to exhibit partially populated secondary structures within some segments; these nascent structures have been termed intrinsically folded structural units (IFSUs) An N-terminal segment of the Cip/Kip proteins termed the kinase inhibitory domain (KID) has been shown to be necessary and sufficient for inhibition of Cdk/cyclin complexes We previously reported that the most prominent IFSU within the p27-KID is sub-domain LH which exhibits nascent α-helical characteristics before binding to Cdk/cyclin complexes in vitro kinase inhibition assays. The capacity of the mutants to regulate the G1/S phase transition was investigated by performing cell cycle arrest assays with NIH 3T3 mouse fibroblast cells transduced with retroviruses encoding the p27 variants. Together, the results of these experiments provide novel insights into the role of nascent helicity within the LH sub-domain in mediating the kinase inhibitory function of p27.In the current study, we have tested this hypothesis by preparing p27-KID variants with either a more or less helical LH sub-domain and studying their interactions with Cdk2/Cyclin A. We also prepared a third p27-KID variant in which the LH sub-domain was replaced with a loop which lacked a sequence relation to the Cip/Kip proteins. The structure of the mutants was analyzed by circular dichroism spectropolarimetry (CD) and the thermodynamics of their interaction with Cdk2/Cyclin A with isothermal titration calorimetry (ITC). CD was also used to determine the thermal stability of the ternary complexes formed by the mutants on binding Cdk2/Cyclin A. In addition, we determined the Cdk2/Cyclin A inhibition potencies of the p27-KID variants by performing wt, p27-KID+H and p27-KID−H, respectively), and a mutant exhibiting random coil secondary structure in the LH sub-domain (p27-KIDloop). We determined the secondary structure of the soluble proteins by CD and selected three constructs for our studies (222) for the p27-KIDwt, p27-KID+H, p27-KID−H, and p27-KIDloop were −4.0, −5.8, −3.0, and −2.1, respectively. p27-KID+H and p27-KID−H were more and less helical than p27-KIDwt, respectively. The p27-KIDloop variant exhibited features of a complete random coil, with little ellipticity at 222 nm and a minimum at 200 nm. We previously showed that the weak helical ellipticity at 222 nm for p27-KIDwt could be attributed to the LH sub-domain We designed (as detailed in File S1), expressed, and purified a number of p27-KID variants in order to obtain mutants that were more or less helical than the wild-type KID construct  for p27-KIDwt, p27-KID+H, p27-KID−H, and p27-KIDloop were −43.8±1.4 Kcal mol−1, −32.7±2.6 Kcal mol−1, −30.5±0.8 Kcal mol−1, and −29.1±1.2 Kcal mol−1, respectively −ΔG (p27-KIDwt)], demonstrating that increasing the helical content of the linker domain did not make the interaction of p27-KID with the Cdk2/cyclin A complex more favorable; in fact, it actually caused it to be slightly less favorable. The reduced helical content of p27-KID−H did not affect the ΔG of interaction with Cdk2/cyclin A based on the finding that the ΔG values for p27-KIDwt and p27-KID−H were identical within error. The thermodynamics of the interaction of the p27-KIDloop mutant with Cdk2/cyclin A, highlights the significance of the nascent helicity of p27-KID. Replacing the native LH sub-domain with a loop composed of residues with a low helical propensity made the interaction with Cdk2/cyclin A less favorable than with the wild-type sub-domain; the ΔΔG value for p27-KIDloop with respect to p27-KIDwt was +2.2 Kcal mol−1. As previously reported loop binding to Cdk2/cyclin A show that disruption of the nascent helicity of the LH sub-domain is thermodynamically unfavorable. In summary, the results of our thermodynamic studies show that perturbation of the helical features of the LH sub-domain affected the thermodynamics of the interaction of p27-KID with the Cdk2/cyclin A complex.These enthalpies of interaction, for the wild-type p27-KID and the variants, exceeded the respective unfavorable interaction entropies and resulted in overall favorable Gibbs free energies of interaction (ΔG). ΔG values of −11.6±0.3 Kcal molmapp) of 54.7±0.6°C wt binds to the Cdk2/cyclin A complex, the thermal stability of the ternary complex formed was greatly increased to 82.3±0.5°C. The Tmapp values for the p27-KID+H, p27-KID−H, and p27-KIDloop ternary complexes were 80.3±0.2°C, 78.5±0.6°C, and 73.0±0.3°C respectively (−H) or completely abolishing the nascent helical character (p27-KIDloop) had a larger effect on thermal stability when compared to increasing the helical content (p27-KID+H). The observation of destabilization of the ternary complex formed by the p27-KID+H variant suggested that the flexibility of the linker domain may play a significant role in the folding of the p27-KIDwt on binding to the Cdk2/cyclin A complex to form a thermally stable ternary complex. On the other hand, the Tmapp values obtained for the p27-KID−H and p27-KIDloop mutants suggest that excessive flexibility of the linker domain is also destabilizing.The Cdk2/cyclin A complex exhibits modest thermal stability, with an apparent melting temperature . These data suggested that the Cdk binding domain and other portions of the KID (the cyclin binding and LH sub-domains) are required for complete and potent Cdk inhibition. However, the specific role of the linker domain in the mechanism of Cdk2/cyclin A inhibition by the KID is not understood.It has previously been shown that p27-KID is a potent inhibitor of the kinase activity of the Cdk2/cyclin A complex 50 values that were greater than the true values. All of the sub-domain LH variants inhibited kinase activity in the low nanomolar range. However, none of the variants were more potent than the wild-type KID. The IC50 values for p27-KIDwt, p27-KID+H, p27-KID−H, and p27-KIDloop were 220 pM, 4.1 nM, 290 pM, and 16.0 nM, respectively  did not diminish the inhibition potency of the KID; we observed that the p27-KID−H variant was almost as potent as the wild-type KID. In summary, while increasing the helical content of sub-domain LH reduced the Cdk2/cyclin A inhibition potency of p27-KID, decreasing the helical content did not have a significant effect. In contrast, abolishing the nascent helicity of sub-domain LH (p27-KIDloop) greatly reduced the inhibition potency.For kinase inhibition assays, a very low concentration of the Cdk2/cyclin A kinase complex (80 pM per reaction) was used to prevent inhibitor depletion which would otherwise result in ICectively . The reset. al, discovered p27 as a potent inhibitor of Cdk2 complexes during the G1 phase of the cell cycle wt, p27+H, p27−H, and p27loop. The retroviruses were used to transduce NIH 3T3 mouse fibroblast cells and the fraction of cells in S phase was used as a measure of cell cycle arrest at the G1 to S transition  of cells observed in S phase for the p27−H mutant. The cell cycle effects of replacing the LH sub-domain with a loop (p27loop) were similar to those observed due to destabilizing the LH sub-domain (p27−H), with 30.5±1.2% of cells observed in S phase for the p27loop variant. The cell cycle analysis of the capacity of the p27 variants to cause cell cycle arrest showed that reduction or elimination of the nascent helicity of sub-domain LH had dramatic effects on the cell cycle arrest capacity of p27; however, increasing the helical content did not have a significant effect.Polyak, ansition . The ret+H), decreased (p27-KID−H), or completely eliminated (p27-KIDloop), with Cdk2/cyclin A. The original rationale for these experiments was the past observation  corresponded to a reduced binding-associated entropy change in comparison with that observed for p27-KIDwt . The value of –TΔΔS for this pair of reactions was −9.9 kcal mol−1 [−TΔΔS = [−TΔS (p27-KID+H)] – [−TΔS (p27-KIDwt)]]. However, examination of other thermodynamic parameters associated with this binding reaction indicated that the favorable enthalpy of binding was reduced by an amount that was slightly greater in absolute value than the reduction in entropic penalty [ΔΔH  =  ΔH (p27-KID+H)−ΔH (p27-KIDwt) = +11.1 kcal mol−1]. This approximate enthalpy/entropy compensation probably arose from incomplete folding of the variant LH sub-domain of the p27-KID+H construct upon binding to Cdk2/cyclin A, as was observed previously for another series of LH sub-domain variants −H variant gave rise to an overall ΔG value that was essentially identical to that observed for p27-KIDwt. This result indicates that nascent helicity within the LH sub-domain and extensive folding upon binding of this sub-domain are not requirements for tight binding to Cdk2/cyclin A. However, the highly disordered features of the p27-KIDloop variant were associated with less favorable binding to Cdk2/cyclin A than for the other constructs, suggesting that intrinsic features of the LH sub-domain of the p27-KID−H variant, apart from its heightened disorder, are favorable for binding. Results from another recent study by Wang, et al., −H is enriched in glutamic acid residues while that of p27-KIDloop is depleted in these, possibly partly explaining the differences observed in their respective thermodynamic binding parameters. In summary, modulating the nascent helicity of the LH sub-domain of p27 did not give rise to the changes in binding energy (ΔG) predicted based on a simple model considering only the change in entropy of this sub-domain upon binding to Cdk2/cyclin A. Our findings suggest that features of the natural p27 LH sub-domain that were associated with maximal binding enthalpy and presumably maximal folding upon binding were not captured in the variant LH sub-domain constructs. In contrast, however, two of these variants did exhibit relatively tight binding to Cdk2/cyclin A , indicating that extensive folding upon binding (within the LH sub-domain for these constructs) is not a requirement for disordered protein/folded protein interactions. This latter observation is consistent with recent observations on persistent disorder within the dynamic complex formed by Sic1 and Cdc4 At first, it seemed as though the binding entropy data for p27-KIDin vitro assays showed that the p27-KID LH sub-domain variants exhibited rank order behavior similar to that observed for ΔG values determined using ITC. For example, while all p27 LH sub-domain variants caused stabilization, p27-KIDwt and p27-KID−H stabilized their complexes with Cdk2/cyclin A to the greatest extent and the p27-KIDloop variant to the smallest extent. This rank order behavior was recapitulated in the results from the in vitro Cdk2 inhibition assays. In these assays, p27-KIDwt and p27-KID−H were the most potent inhibitors of Cdk2/cyclin A, with p27-KID+H and p27-KIDloop>10-fold and >50-fold less potent, respectively, than the former two constructs. Collectively, our ITC, thermal stabilization and in vitro biochemical results support the view that alteration of the LH sub-domain within the kinase inhibitory domain of p27 significantly influences the thermodynamic and functional properties of the full KIDs.Other wt) and more helical (p27+H) LH sub-domains were equally potent in causing arrest in G1 phase of the cell division cycle. The result for p27+H is in contrast to the reduced activity of the corresponding KID construct in the in vitro assays. The two other full-length LH sub-domain variants, p27−H and p27loop, caused essentially no reduction in the fraction of cells in S phase but were associated with an increased G0/G1 content (relative to the GFP only control), indicative of partial cell cycle arrest. These results may reflect differential interactions of the various p27 LH sub-domain variants with the repertoire of Cdk/cyclin complexes which control entry into and progression of the cell division cycle from G0 to G1 and then to S phase. Alteration of the ability of the related Cdk inhibitor, p21, to promiscuously interact with and inhibit multiple Cdk/cyclin complexes (through lengthening or shortening of the LH sub-domain) was previously shown to alter the ability to cause complete cell cycle arrest −H and p27loop LH sub-domain variants exhibit altered Cdk/cyclin binding promiscuity, which is manifested as incomplete cell cycle arrest.The biological consequences of LH sub-domain alteration were probed by determining the cell cycle arrest properties of the corresponding full-length p27 variants . The p27We observed a dependence of the ability of the KID of the Cip/Kip proteins to interact with a Cdk/cyclin complex on the features of the so-called “linker” or LH sub-domain even though it has been reported that this sub-domain does not significantly contribute to the favorable thermodynamics of interactions with Cdk2/cyclin A Our approach of designing p27-KID mutants with linker domains that were either more or less helical than the wild-type p27-KID was to substitute solvent exposed residues within the linker domain with residues that are known to either stabilize or destabilize helices. Mutating the solvent exposed residues minimizes disruption of contacts between p27 and the Cdk2/cyclin A complex. This reduced the possibility that the results obtained from the succeeding studies were a consequence of the disruption of contacts between p27 and Cdk2/cyclin A. The solvent accessible surface area (SASA) for each residue within the linker domain was analyzed using the GETAREA program wt protein was expressed and purified as previously described +H ) or GST-6x-His fusion constructs (p27-KID−H and p27-KIDloop) and expressed in Escherichia coli BL21 (DE3) cells . The proteins were purified by nickel affinity chromatography and then digested with thrombin to remove the fusion tags. This was followed by boiling for 20 minutes and separation of the supernatant (purified protein) and the pellet by centrifugation. Full length human Cdk2 phosphorylated at threonine 160 (Cdk2) and truncated human cyclin A (residues 173–432) were expressed and purified using established protocols wt or the LH sub-domain variants with purified Cdk2/cyclin A complex in a 1.1∶1.0 ratio, incubating the mixture for 30 minutes and purifying the complexes by gel filtration chromatography . Prior to the ITC experiments or ternary complex preparation, p27-KIDwt and its variants were treated with 100 mM DTT at room temperature for 30 minutes followed by gel filtration chromatography purification. Protein concentrations were determined by UV absorbance at 280 nm as detailed in File S1.p27-KIDwt or its LH variants (in the syringe) were titrated into Cdk2/cyclin A complex (in the cell). The titration sequence included a single 2 µL injection followed by 39 injections, 6 µL each, with a spacing of 300 seconds between the injections. The protein concentrations were as follows: 75 µM p27-KIDwt into 7.4 µM Cdk2/cyclin A; 56 µM p27-KID+H into 4.2 µM Cdk2/cyclin A; 42 µM p27-KID−H into 4.4 µM Cdk2/cyclin A; and 36.4 µM p27-KIDloop into 3.1 µM Cdk2/cyclin A. OriginLab software .) was used to fit the raw data to a 1∶1 binding model.ITC experiments were done in a buffer composed of 20 mM HEPES pH 7.5, 300 mM NaCl, and 5 mM DTT, at 25°C using a VP-ITC calorimeter . p27-KIDwt or its variants interacted with Cdk2/cyclin A was determined using an AVIV model 62A DS circular dichroism spectropolarimeter equipped with a thermoelectric temperature controller . Each ternary complex sample contained 400 nM of protein in 1 mM sodium phosphate buffer pH 7, 25 mM NaCl and 1 mM DTT. Denaturation was monitored by ellipticity at 222 nm in the temperature range of 15°C to 95°C. Ellipticity readings were taken every 1°C at a heating rate of 5°C min−1 in a 1 cm path length quartz cuvette with constant stirring. Each experiment was performed in triplicate. The curves were fit as previously described mapp). Apparent thermal denaturation temperatures are reported because the denaturation process is irreversible due to protein precipitation after denaturation.The thermal stability of the ternary complexes formed when p27-KIDwt or its mutants and incubated at 4°C for 60 minutes. This was followed by addition of 6 µCi γ 32P-ATP  to each reaction and incubation at 30°C for 35 minutes. Each reaction had a total volume of 15 µL. The sample buffer contained 20 mM HEPES pH 7.3, 25 mM sodium β-glycerolphosphate, 15 mM MgCl2, 16 mM EGTA, 0.5 mM Na3VO4 and 10 mM DTT. The reactions were stopped by adding SDS-PAGE gel loading buffer and were then analyzed by SDS-PAGE. A phosphoimager  was used to quantify the bands on the gel and data fitting was done using the Graphpad Prism software . Each curve was fit to a triplicate data set to derive values of p27-KID (or p27-KID variant) concentration that caused 50% inhibition  of the kinase complex and the errors are reported as the 95% confidence interval.Cdk2/cyclin A kinase (80 pM) was mixed with 2.5 µM histone H1 (Upstate) and varying quantities of p27-KIDThe coding sequences for full-length versions of human p27 containing either the wild-type LH sub-domain or the variants, additionally with a C-terminal HA tag, were sub-cloned into an MSCV packaging vector and these vectors were used for preparation of packaged viruses that expressed HA-tagged wild-type and the LH sub-domain variants of full-length p27 and GFP 6 cells were cultured on each plate. After 24 hours, the cell culture media (with additives) was substituted with additive free media (DMEM only) and incubated for 3 hours. During this time, transfection cocktails were prepared.Retroviruses encoding HA tagged full-length constructs of the p27 variants were packaged in 293T cells. 100 mm×10 mm plates of 293T cells were cultured at 37°C in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2% of 200 mM glutamine (vol:vol), and 1% of 10,000 µg/µl penicillin/streptomycin (vol:vol). Hereafter, the DMEM supplements are referred to as additives. 3×10The cocktails contained a single helper DNA , shuttle vector DNA plasmid, fugene 6 transfection reagent  and additive free DMEM media. The helper DNA was obtained from Dr. Martine Roussel . In every cocktail, the ratio of the volume of the fugene reagent to the amount of the shuttle vector (by mass) was varied to identify the ratio that yielded the highest viral titre. The volume of the fugene reagent was kept constant at 15 µl and the amount of the shuttle vector added was varied to give different fugene to shuttle vector ratios. Cocktails with volume of fugene to mass of shuttle plasmid DNA ratios of 1∶20, 1∶15, 1∶12, and 1∶10 were prepared for each construct (each construct had 4 cocktails). A constant 6 µg of helper DNA was added to each coctktail. Serum free media was added to bring the total volume of each cocktail to 700 µl. Before addition of fugene to the cocktails, it was first warmed to room temperature according to manufacturer’s instructions. For each cocktail, the reagents were added in the following order: (i) serum free media, (ii) helper DNA, (iii) vector DNA, and (iv) fugene. The cocktails were then mixed. After 3 hours, the transfection cocktails were added to the incubated cells.After 20 hours the additive free media was replaced with 10 ml of fresh media supplemented with additives. In the following steps, all the media was supplemented with additives unless stated otherwise. After 6 hours, the media was aspirated from the plates and 5 ml of media was added to each plate. After 18 hours, 5 ml of the media containing virus was harvested and 5 ml of fresh media was added to each plate. 8 hours later, 5 ml of media containing virus was again harvested and a fresh 7 ml of fresh media added to each plate.Virus-containing media (7 ml) was harvested a final time after an additional 20 hours. The harvested virus was filtered using a 0.22 µm steriflip filter  and stored at 4°C.5 NIH 3T3 mouse embryo fibroblast cells were cultured on 100×10 mm plates in DMEM media with additives (additives specified in section 2.9 above). After 20 hours, the media was aspirated from each plate. 5 ml of cold virus (virus kept in ice) supplemented with 30 µl of 1 mg/ml of polybrene (6 µl of polybrene for every ml of virus) was added to each plate and incubated at 37°C. After 3 hours, 10 ml of DMEM media with additives was added to each plate. After 48 hours, the cells were washed, re-suspended in additive-free DMEM media, and the level of protein expression determined using GFP fluorescence analysis . Before GFP analysis, viable cells were separated from non-viable cells by propidium iodide (PI) staining. For PI staining, the cells were first mixed until a single suspension of cells was realized. The cells were then counted and washed once with phosphate buffered saline (PBS). The supernatant was decanted and the cells were resuspended in PI solution (0.05 mg/ml propidium iodide) at 1×106 cells/ml. This was followed by treatment with RNAse for 30 minutes at room temperature. Prior to and after the RNAse step, the cells were kept in ice. The cells were then filtered using a 40 µm-diameter mesh  to eliminate large multicellular clumps that might clog the flow cytometer. The filtered cells were analyzed by flow cytometry.3×10For cell cycle analysis, viral solutions with high viral titres, as determined by GFP analysis, were used to transduce NIH 3T3 cells as described above. The cells were washed, and analyzed for viral transduction using GFP analysis. The top 10% GFP positive cells were stained by PI and analyzed for DNA content. Each cell cycle arrest analysis was performed in triplicate for each construct.Figure S14-H1) by Cdk2/cyclin A in the presence of increasing concentrations of p27-KIDwt or the LH sub-domain variants.Raw data for Cdk2/cyclin A inhibition assays. Phosphorimager analysis of SDS-PAGE results of phosphorylation of Histone H1 (PO(TIF)Click here for additional data file.Table S1Solvent exposure of the residues in the linker domain of p27.(DOCX)Click here for additional data file.Table S2Linker domain sequences for the p27-KID variants designed to be more and less helical.(DOCX)Click here for additional data file.File S1(DOCX)Click here for additional data file.