Solution structure of the active-centre mutant I14A of the histidine- containing phosphocarrier protein from Staphylococcus carnosus Andreas Mo¨ glich 1, *, Brigitte Koch 2 , Wolfram Gronwald 1 , Wolfgang Hengstenberg 2 , Eike Brunner 1 and Hans Robert Kalbitzer 1 1 Institute of Biophysics and Physical Biochemistry, University of Regensburg, Germany; 2 SG Physiology of Microorganisms, Ruhr-University of Bochum, Germany High-pressure NMR experiments performed on the histi- dine-containing phosphocar rier protein (HPr) from Sta- phylococcus carnosus have shown that residue Ile14, which is located in t he active-centre loop, exhibits a peculiarly small pressure response. In contrast, the rest of the loop shows strong pre ssure effects a s i s e xpected for t ypical protein interaction sites. To elucidate the structural role of t his residue, the mutant protein HPr(I14A), in which Ile14 is replaced by Ala, was produced and s tudied by solution NMR spectroscopy. On the basis of 1406 structural restraints including 20 directly detected hydrogen bonds, 49 1 H N - 15 N, and 25 1 H N - 1 H a residual dipolar couplings, a well resolved three-dimensional structure could be determined. The o verall fold of the protein is not influenced by the mutation but characteristic conformational changes are introduced into the active-centre loop. They lead to a dis- placement of the ring system of His15 and a distortion of the N-terminus of the first helix, which supports the histidine ring. In ad dition, the C -terminal helix is bent because the side chain of Leu86 located at the end of this helix partly fills the hydrophobic cavity created by the mutation. Xenon, which is known to occupy hydrophobic cavities, causes a partial reversal of the mutation-induced structural effects. The observed structural changes explain the reduced phospho- carrier activity of the mutant and agree well with the earlier suggestion that Ile14 r epresents an anchoring point stabil- izing the active-centre loop in its correct conformation. Keywords: histidine-containing phosphocarrier protein (HPr); mutant protein; nuclear magnetic resonance (NMR); protein structure. Histidine-containing phosphocarrier protein (HPr) is a central part of the bacterial carbohydrate/phosphoenolpyru- vate phosphotransfer system (PTS) first described in Escheri- chia coli [1]. The PTS catalyses the phosphorylation of a metabolite and its concomitant transport across the plasma membrane into the cytosol (PTS reviewed in [2,3]). During the transport p rocess, the phosphoryl group of phos- phoenolpyruvate is transferred first to enzyme I (EI) and then to His15 of HPr. The phosphoryl group is transiently bound to the N d1 atom of the imidazole ring of His15. Via enzymes IIA, IIB and IIC/D, the group is finally transferred to the imported metabolite. Compared to conventional substrate import and consecutive phosphorylation, the import v ia the PTS is energetically favorable. From the residue His15 of HPr, the phosphoryl group can also be transferred t o transcription factors containing PTS regula- tion domains (PRDs). Depending on their phosphorylation state, these proteins control the activity of operons mainly responsible for catabolism [3,4]. The activity of HPr from Gram-positive bacteria is regulated by the bifunctional enzyme HPr kinase/phosphorylase, which controls the phosphorylation state of the HPr residue Ser46 [5]. When phosphorylated at residue Ser46, HPr interacts with cata- bolite control protein A (CcpA), which regulates the activity of genes involved in carbon and nitrogen metabolism [6,7]. To exert its various biological functions, the HPr molecule must be able to interact with different proteins and ligands in a tightly regulated manner mainly depending upon the nutritional state of the bacterium. Probably these different interactions are mediated by conformational changes of HPr, particularly in the active site region. Structural studies have contributed significantly to a detailed understanding of the bacterial PTS. The three-dimensional structu res of HPr molecules from different organisms have been determined both by NMR spectroscopy and X-ray crystallography (e.g. [8–12]). Signi- ficant structural changes in the active site region of the HPr were observed upon phosphorylation at Ser46 [13,14]. The solution structure of HPr from Staphylococcus carnosus,a small protein with a molecular mass of 9511 Da, has been determined by Go ¨ rler et al. [15]. It shows the open-faced b-sandwich fold common to all HPr structu res known so f ar. It consists of a four-stranded antiparallel b-sheet, one short Correspondence to H. R. Kalbitzer, Institute of Biophysics and Phys- ical Biochemistry, University of Regensburg, Regensburg, Germany. Fax: +49 941943 2479, Tel.: +49 941943 2594, E-mail: hans-robert.kalbitzer@biologie.uni-regensburg.de Abbreviations: CHAPSO, 3-( cholamidopropyl)-dimethylammonio 2-hydroxyl-1-propane sulfonate; DIODPC, 1,2-di-O-dodecyl-sn-glyc- ero-3-phosphocholine; HPr, histidine-containing phosphocarrier pro- tein; PRD, PTS regulation domain; PTS, phosphoenolpyruvate- dependentphosphotransferasesystem;RDC,residualdipolarcoupling. *Present address:DepartmentofBiophysicalChemistry,Biozentrum, University of Basel, Switz erland. (Received 28 July 2004, revised 13 October 2004, accepted 21 October 2004) Eur. J. Biochem. 271, 4815–4824 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04447.x and t wo long a-helice s. K albitzer et al.[16]usedhigh- pressure 1 Hand 15 N N MR measurements to study the ability of HPr to adopt different conformations. In general, dynamic regions in proteins are expected to be capable of undergoing large conformational changes. This should result in strong effects induced by the variation of external conditions such as pressure. While the core region of HPr from S. carnosus, w hich mainly consists of the b-sheet, showed only little or m oderate variation, large pressure- induced changes o f chemical shifts w ere observed i n the active site region encompassing residues 12–18. In contrast to its surrounding residues, Ile14 displayed only a s mall pressure-induced change of chemical shift. Kalbitzer et al. [16] suggested that this r esidue, which is not strictly conserved between different species, serves as an anchoring point for the active site loop. The isoleucine side chain would stabilize the loop but still allow it to adopt the different conformations necessary for the interaction with diverse proteins andligands. To evaluate this hypothesis, a mutant form of HPr from S. carnosus was produced in which the isoleucine at position 14 is replaced by an alanine residue, referred to as HPr(I14A). In this paper we report the solution structure of this protein as determined by NMR spectroscopy. Materials and methods Protein purification and sample preparation The gene for the I14A mutant of HPr was constructed using the PCR-Megaprimer method [17] and cloned into the pET11 vector. T he plasmid was overexpress ed in E. coli strain BL21 DE3. HPr(I14A) was isolated as described previously [18]. Uniformly 15 Nand 15 N/ 13 C isotope-labelled protein was obtained accordingly. HPr(I14A) was studied under the same conditions as used for the structure determination of the wild-type protein [15]. Lyophilized HPr protein was dissolved in a buffer solution containing 20 m M potassium phosphate, 100 m M KCl, 0.1 m M EDTA, 1m M NaN 3 ,1 l M pepstatin, 1 l M leupeptin, 0.1 l M bovine pancreatic trypsin inhibitor and, as an internal reference, 0.1 m M 2,2-dimethyl-2-silapentanesulfonic acid. The pH was adjusted to 7.14 by addition of KOH. The fi nal protein concentrationwasbetween1.4and1.7m M depending on the NMR experiment. For the determination of residual dipolar couplings (RDCs), partial molecular orientation of the HPr sample was obtained by the addition of 7.5% or 4.0% (w/v) of the bicelle forming lipid mixture 1,2-di-O- dodecyl-sn-glycero-3-phosphocholine (DIODPC)/3-(cho- lamidopropyl)-dimethylammonio 2-hydroxyl-1-propane sulfonate (CHAPSO) at a 4.3 : 1 ratio [19]. NMR spectroscopy NMR spectra were recorded on Bruker (Karlsruhe, Ger- many) DMX-500, DMX-600 and DMX-800 spectromete rs with 1 H resonance frequencies of 500, 600 and 800 MHz, respectively. All measurements were carried out at a temperature of 2 98 K. Time-domain NMR data were processed using the XWINNMR package (Bruker). P roton chemical shifts were assigned on the basis of 2D TOCSY and HCCH-TOCSY spectra measured a t 500 and 600 M Hz, respectively. Nitrogen ( 15 N) and carbon ( 13 C) resonances could be d etermined from HSQC, HNCA, HNCO and CBCA(CO)NH spectra recorded at 600 MHz. Distance restraints were derived from homonuclear 2D NOESY spectra in 1 H 2 Oand 2 H 2 O and from a 13 Cresolved NOESY spectrum, measured at 800, 500 and 600 MHz, respectively. The assignment of NOE signals was facilitated by using a homology structure of the HPr(I14A) protein which was generated by the computer program PERMOL (A. Mo ¨ glich, D. Weinfurtner, T. Maurer, W. Gronwald, H. R. Kalbitzer, unpublished data). From this structure and the assigned resonance frequencies, 2D NOESY spectra were calculated using the computer program RELAX [20]. These calculated spectra w ere c ompared with the experimental data. The 1 H chemical shifts were referenced relative to 2,2- dimethyl-2-silapentane-5-sulfonic acid. 15 Nand 13 Creso- nances were referenced indirectly [21]. Spectral visualization and volume i ntegration of NOE signals was carried out using the computer program AUREMOL [22]. Determination of dihedral angles and hydrogen bonds Three-bond coupling c onstants between H N and H d atoms, 3 J HN-Ha , were measured by MOCCA-SIAM experiments [23,24]. T he values of the coupling constants were determined using the procedure described by Titman and Keeler [25]. Structural restraints for the main chain dihedral angles F were calculated according to the Karplus equation [26] employing the parameters determined by Vuister and Bax [27]. Hydrogen bonds between main chain amide protons and carbonyl oxygen atoms were directly detected in H(N)CO experiments as described by Cordier et al. [28,29]. Residual dipolar couplings The measurement of residual d ipolar couplings requires partial molecular a lignment of the sample molecules, which was obtained by t he addition of 7.5% (w/v) of the DIODPC/ CHAPSO lipid mixture to the sample solution. RDCs [30–32], were measured for the 1 H N - 15 N amide bond using both conventional nondecoupled 1 H- 15 N-HSQC and IPAP- [ 1 H- 15 N]-HSQC experiments [33]. R DCs were de termined as the difference between the coupling constants observed in isotropic and anisotropic solution. Using the computer programs SVD [34], DIPOCOUP [35] and PALES [36], the molecular alignment tensor could be determined from the measured residual dipolar couplings and a structure model that was calculated from all experimental restraints except for the residual dipolar couplings. The eigenvalues of the tensor were found to be S zz ¼ 0.000491, S yy ¼ )0.000313, S xx ¼ )0.000178. From the experimental residual dipolar couplings and the alignment tensor, the quality factor Q can be calculated [37]. A value of 0.2880 was obtained, indicating good agreement between the RDCs and the other structural restraints derived from NMR experiments. MOCCA-SIAM experiments in isotropic and anisotropic solution were used to measure residual dipolar couplings for the 1 H N - 1 H a -coupling. In this case the anisotropic solution contained only 4.0% (w/v) of the above lipid mixture. The values for the residual dipolar couplings were again determined as the difference of the coupling constants in isotropic and anisotropic solution. At pH values above 6.0, Cavagnero et al. [19] reported severe line broadening of 4816 A. Mo ¨ glich et al.(Eur. J. Biochem. 271) Ó FEBS 2004 resonance lines in solutions containing the DIODPC/ CHAPSO lipid mixture, which was ascribed to aggregation of the lipid bicelles. Due to this effect (which also occurred in this study) only a limited number of 1 H N - 1 H a -RDCs could be determined with sufficient accuracy. Structure calculation Structures were determined by simulated annealing employ- ing version 1.0 of the computer program CNS [38,39]. A total number of 1406 non redundant structural restraints derived from NMR experiments were used in the calculations (Table 1). This corresponds to a ratio of about 16 restraints per residue. Approximate distances between 1 Hatomswere derived from NOE cross-peak intensities in two- and three- dimensional spectra. The standard simulated a nnealing protocol supplied with CNS was modified to allow two different classes of residual dipolar couplings to be used as restraints. Apart from this, all oth er p arameters correspon- ded to the standard values. Of 300 calculated structures, the ensemble of the 10 structures with the lowest pseudoenergies was further refined in explicit solvent [40,41]. To facilitate comparison with the wild-type HPr from S. carnosus its structure was recalculated employing exactly the same protocol as for the mutant protein. Mean structures of the mutant and wild-type HPr proteins were calculated with the computer program MOLMOL [42] by fitting the positions of the backbone atoms C a ,C¢ andN.Structuralimageswere prepared with the computer program MOLMOL and rendered with POVRAY (http://www.povray.org). Database deposition Chemical shift values for 1 H, 13 Cand 15 N atoms have been deposited in the BioMagRes database (entry number 6254), and the atomic coordinates of the structure in the Protein Data Bank under PDB accession code 1TXE. Calculation of combined chemical shift changes HPr(I14A) was investigated in the presence [43] and absence (this study) of xenon. The combined chemical shift changes Dd tot between two states a and b are calculated from the amide proton shifts, d H , and the amide nitrogen shifts, d N ,in these two states according to Eqn (1 ): Dd tot ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi w 2 ðd a H À d b H Þ 2 þðd a N À d b N Þ 2 q ð1Þ Here, w denotes a weight factor that accounts for the different s ensitivities of the c hemical s hifts of the amide proton and the amide nitrogen towards structural changes and xenon, respectively. Following Gro ¨ ger et al .[43],w was computed as the ratio of the s tandard deviations of the chemical shifts of the amide nitrogen and p roton nuclei. Results Determination of the three-dimensional structure of HPr(I14A) To allow the comparison of the structures of HPr(I14A) and the wild-type protein the same experimental conditions were used as in the study of Go ¨ rler et al. [15]. Spectral assign- ments of 1 H, 13 Cand 15 N resonance lines have been obtained from conventional homonuclear and h etero- nuclear NMR e xperiments. A list of the restraints used in Table 1. Structural statistics of H Pr(I14A). NMR experiments were conducted at 298 K and pH 7.14. Structures were calculated with CNS using the standard simulated annealing pro tocol including the use of two different classes of residual dipolar couplings [38,39], followed by a refinement in explicit solvent [40,41]. The quality of the 10 lowest energy structures was assessed using PROCHECK - NMR [46]. Type of restraint Number of restraints NOE contacts 1268 Intraresidual (i, i) 637 Short and intermediate distance (i, i + j; 1 £ j £ 4) 365 Long distance (i, i + j; j ‡ 5) 266 F dihedral angles from 3 J couplings (MOCCA-SIAM) 44 Hydrogen bonds from H(N)CO experiment 20 RDCs 74 1 H N - 15 N RDC from IPAP/HSQC experiments 49 1 H N - 1 H a RDC from MOCCA-SIAM experiments 25 Total 1406 Quality factors for the residual dipolar couplings Q 1 H N - 15 N residual dipolar couplings 0.2880 1 H N - 1 H a ´ RDC residual dipolar couplings 0.4089 Restraint violations in the 10 lowest-energy structures number NOE violations > 0.05 nm 0 J-coupling violations > 1.7 Hz 6 RMSD values for the 10 lowest-energy structures RMSD (nm) Core region (residues 2–9, 16–27, 32–37, 40–43, 47–53, 59–84), backbone atoms C a ,C¢,N 0.066 Core region (residues 2–9, 16–27, 32–37, 40–43, 47–53, 59–84), heavy atoms 0.102 All residues, backbone atoms C a ,C¢, N 0.084 All residues, heavy atoms 0.121 Ramachandran plot (except glycine and proline residues) Incidence Most favored regions 83.7% Additional allowed regions 13.5% Generously allowed regions 1.7% Disallowed regions 1.1% Energies of the 10 selected structures after refinement in water E/kJÆmol )1 E total )13534 ± 306 E NOE 137 ± 11 Ó FEBS 2004 Solution structure of the I14A mutant of HPr (Eur. J. Biochem. 271) 4817 the structure calculation of H Pr(I14A) is given in Table 1. Interatomic distance restraints have been derived from homonuclear and 13 C-resolved NOESY spectra. Structural restraints for the backbone dihedral angle F were calculated from three bond J-couplings between H N and H a atoms measured in MOCCA-SIAM spectra [24]. Twenty hydro- gen bonds could be directly detected in H(N)CO experi- ments [28,29]. Using nondecoupled 1 H- 15 N-HSQC and IPAP-[ 1 H- 15 N]- HSQC experiments [33], RDCs for the 1 H N - 15 N bond vector were determined. Remarkably, these RDCs showed a bimodal frequency distribution in contrast to the unimodal distribution expected for isotropically distributed bond vectors [31]. A comparison of the sequence dependence of the observed RDCs with a prediction of secondary structure based upon chemical shift values [44] shows that the size of the c oupling is strongly dependent upon the secondary structure ( Fig. 1). Residues in a-helices mainly display positive 1 H N - 15 N RDCs while those located in b-sheets usually show negative values. This observation can be accounted for by the orientation of the principal axis system of the molecular alignment tensor in the HPr molecule (Fig. 2). The z-axis, which denotes the direction o f largest partial molecular orientation, is arranged almost parallel to the a-helices and the b-sheet of the protein. As the 1 H N - 15 N bond vectors in the a-helices are therefore almost parallel to the z-axis, their size is mainly determined by the positive eigenvalue S zz of the tensor [31]. In contrast, the 1 H N - 15 N bond vectors in the b-sheets are almost perpendicular to the z-axis and, therefo re are determined by the negative eigenvalues S yy and S xx . A similar dependence of the magnitude and sign of residual dipolar couplings on the secondary structure was also reported for the F48W mutant of HPr from E. coli [45]. Residual dipolar couplings for the vectors connecting the H N and H a atoms have been determined in MOCCA-SIAM experiments. Structures were calculated by simulated annealing fol- lowed by further refinement in water. A total of 1406 structural restraints was used, corresponding to a ratio of approximately 1 6 r estraints p er amino acid r esidue (Table 1). A n average st ructure was c alcu lated from the final 10 models (Fig. 3). Figure 2 shows a schematic representation of the secondary structure elements of HPr(I14A). Overall, the structure is well defined but the precision varies in different regions according to the number of experimental restraints, which is indicated by the colour code in Fig. 3. For the heavy (nonhydrogen) atoms and the backbone atoms (C a ,C¢, N) located in the core region of the molecule comprising th e canonical secondary structure elements, RMSD values of 0.102 nm and 0.066 nm were obtained, respectively (Table 1). Larger variations can be seen in the region of the loops L1 con necting strand A with helix a, and L5 joining helix b with strand D. HPr(I14A) shows the open-faced b-sandwich fold which was also observed for the wild-type protein and other HPr molecules. The analysis of the 10 lowest energy structures with the Fig. 1. Dependen ce of 1 H N - 15 N residual dipolar couplings on secondary structure. Thesizeofthe 1 H N - 15 N residual dipolar couplings is strongly correlated with secondary structure. A prediction of sec ondary struc- ture elements by the program CSI [44] based upon the chemical shift values of the H a ,C a ,C b and C¢ atoms is shown in grey. The observed 1 H N - 15 N residual dipolar couplings are plotted as a function of amino acid number, shown in white. A clear correlation between secondary structure and the magnitude of the coupling values can be seen with residues in a-helical regions predominantly showing positive residual dipolar couplings and residues in b-sheet regions having negative values. Note that a residual dipolar coupling of )23 Hz has been measured for residue 60, which is located in a loop region. For sake of clarity the ordinate of th e figure has bee n restricted t o the regio n of )15 to 15 Hz . Fig. 2. Three-dimensional structure of HPr(I14A) relative to molecular alignment t ensor. The 3D structure of HPr(I14A) is shown relative to the principal axis system of the molecular alignment tensor d etermined for the 1 H N - 15 N residual dipolar couplings. The se condary structural elements are indicated by labels. Note that the z-axis denoting the direction of largest partial alignment is oriented nearly parallel to the b-sheet and the a-helices. The eigenvalues of the tensor are S zz ¼ 0.000491, S yy ¼ )0.000313, S xx ¼ )0.000178. 4818 A. Mo ¨ glich et al.(Eur. J. Biochem. 271) Ó FEBS 2004 program PROCHECK - NMR [46] recognizes a central antipar- allel b-sheet consisting of strands A (residues 2–7), B (31–37), C (40–43) and D (60–67), two relatively long a-helices a (16–27) and c (70–83), as well as the short a-he lix b (47–50). The analysis of chemical shifts [44] predicts essentially the same secondary structure elements at slightly different positions (b-strands: strand A, 1–9; strand B, 32–36; strand C, 39–42; strand D, 59–66; a-helices: helix a, 12–26; helix b, 47–51; helix c, 69–82). The active site of the HPr molecule containing the residue His15 is formed by loop L1. Recalculation of the 3D-structure of wild-type HPr and comparison with the mutant protein It is known that the NMR structures obtained from a given set of experimental restraints also depend on the programs used for the structural calculations. Even when using the same program, they depend on the specific protocol used for the calculations. Therefore, we recalculated the structure of the w ild-type protein on the b asis of the restraints used previously [15] with the same protocol used here for the mutant protein. Compared to the wild-type structure of HPr from S. carnosus stored in the PDB (entry 1QR5) no significant structural changes w ere observed. However, the extended water refinement protocol led to a significant improvement of the general geometry. The structural statistics and the PROCHECK - NMR analysis are summarized in Table 2. The wild-type protein and the mutant form studied in this paper show the same global fold with essentially identical secondary structure elements. However, compared to the wild-type p rotein, h elix b is significantly shorter in the mutant protein and distorted at its C-terminal end. In the core r egion of the protein, which encompasses the canonical secondary structural elements, the average struc- tures of the wild-type and the mutant protein molecule agree reasonably well with an RMSD value for the backbone atoms (C a ,C¢, N) of 0.119 nm. When all backbone atoms of the proteins are taken i nto account, this value increase s to 0.155 nm. Significant deviations between t he two proteins are seen in the active site region where the mutation has been introduced. The replacement of I le14 by Ala causes a slight longitudinal compression of the mutant protein (Fig. 4). At its N-terminal end, helix a displays a kink towards the interior of the protein. The space that in the wild-type molecule is occupied by the large hydrophobic side chain of Fig. 3. Structure ensemble of HPr(I14A). The average structure of the 10 lowest- energy structures out of 300 calculated with CNS is shown. TheradiusofthesplinereflectstheRMSDvaluesoftheC a atom positions. Th e s cale bar indicates a length of 0.2 nm correspo nding to a RMSD value of 0.1 nm. Residues are colour-coded according to the number of restraints used in the stru cture calculations for this amino acid. Light grey indicates 10 or fewer, yellow 11–20, orange 21–40, red more than 40 restraints per residue. Table 2. Structural statistics of wild-type HPr. The structures were recalculated from the data from Go ¨ rler et al. [15] with the same pro- tocol used for the mutant. The NMR data have been recorded at 298 K and pH 7.14. In total 1301 NOE, 78 dihedral angle, and 39 hydrogen bond restraints were used. The quality of the 10 lowest energy structures was assessed using PROCHECK - NMR [46]. Restraint violations in the 10 lowest-energy structures Number NOE violations > 0.05 nm 9 J-coupling violations > 1.7 Hz 28 RMSD values for the 10 lowest-energy structures RMSD (nm) Core region (residues 2–9, 16–27, 32–37, 40–43, 47–53, 59–84), backbone atoms C a ,C¢,N 0.071 Core region (residues 2–9, 16–27, 32–37, 40–43, 47–53, 59–84), heavy atoms 0.112 All residues, backbone atoms C a ,C¢, N 0.088 All residues, heavy atoms 0.130 Ramachandran plot (except glycine and proline residues) Incidence (%) Most favored regions 77.5 Additional allowed regions 16.5 Generously allowed regions 4.7 Disallowed regions 1.3 Energies of the 10 selected structures after refinement in water E/kJÆmol )1 E total ) 11752 ± 456 E NOE 485 ± 21.3 Ó FEBS 2004 Solution structure of the I14A mutant of HPr (Eur. J. Biochem. 271) 4819 the isoleucine residue is instead partly filled by the backbone and s ide chain atoms of Ala19. In addition, the C-terminus folds back onto the core of the protein thereby allowing the side chain of Leu86 to partly fill the hole created by the removal of Ile14. Due to these changes other alterations are induced in the HPr(I14A) molecule. The catalytically active residue His15 is moved closer to the p rotein interior and its orientation relative to the protein core is changed. The loops L1 and L5 show a significantly different conformation. Helix b is distorted at its C-terminal end and the loop L4 at its N-terminal end is bent into another d irection than in the wild-type protein. To allow the hydrophobic side chain of Leu86 to project into the protein core, the orientation of helix c is slightly changed in the mutant form. These changes observed in the mutant protein are also supported by other NMR parameters. For example, NOE contacts between the side chain protons of Leu86 and protons of amino acids Ala14, Val55 and Leu81 are observed, none of which are seen for the wild-type protein. Analysis of the backbone dihedral angles F and Y of mutant and wild-type protein also s upports the observed structural differences (Fig. 5 ). Significant changes in dihedral angles between the two proteins were observed for almost all regions of the molecules. In Fig. 5 the residues for which the difference in dihedral angles exceeds the sum of the errors are indicated by black dots. Particularly for residues 13 and 14 of the active-centre loop, residues 38 and 39 of loop L3 and for residue 54 located in loop L5, distinctly different confor- mations are f ound. In ad dition, the dihedral angles of residue 84 are changed in the mutant protein allowing the C-terminus to bend to the protein core. Effect of xenon-binding on the mutation-induced structural changes It has been shown previously [43] that a xenon atom binds into the hydrophobic cavity of HPr(I14A) that i s created by the replacement of t he bulky Ile14 by an alanine (Fig. 6). P otentially, the binding of xenon inside this cavity could lead to a reversal of the structural changes induced by the m utation because the size of an isoleucine side chain almost exactly corresponds to that of a xenon atom. As 1 H N and 15 N chemical shifts provide a sensitive measure for the local structural environment of the amide bond, 1 H N - 15 N-HSQC spectra were recorded for wild-type and mutant HPr. Following Gro ¨ ger et al. [43], combined chemical shift changes were calculated for the amide groups according to Eqn (1). The changes induced by the mutation of the wild-type protein were compared with the combined chemical s hift changes observed in the I14A m utant upon xenon-binding (Fig. 7). While on average the total cha nges in chemical shifts due to the introduction of the mutation are about four times as large as those i nduced by xenon-binding, they show a similar dependence on the amino acid sequence. Note that not only the magnitudes of the individual shift changes but also that their signs closely correspond. Thus, for most residues the chemical shift changes caused by the mutation were at least partly compensated by the binding of xenon. Discussion Structural basis of the strongly reduced pressure response at position 14 in HPr During the phosphoryl group transfer from enzyme EI to enzyme EII or other proteins, the active centre loop L1 of wild-type HPr has to adapt to different functional states. High-pressure NMR spectroscopy studies have revealed that protein regions, which are able to exist in different conformational (sub)states, often show large, n onlinear pressure reponses [47]. In agreement with these findings such a pressure response was also experimentally observed for loop L1 of wild-type HPr [16]. The sole exception was residue Ile14, which is adjacent to the His15 involved in phosphoryl transfer, and shows only a very small pressure response indicating that its position is stabilized in some way. The NMR structure shows the side chain of this amino acid to be located in a hydrophobic cavity, which might possibly stabilize the conformation of this residue as well as that of the entire loop L1. Fig. 4. Comparison of wild-type and mutant HPr. Comparison of the three-dimensional structures of the mutant (left) and wild-type HPr (right). T he sid e chains o f the catalytically active histidine residue 15 and of residue 14 (isoleucine to alanine) are shown in blue and yellow, respectively. Residues Ala19 and Leu86 are indicated in red. The removal of the isoleucine side c hain in the mutant protein leads to significant structural rearrangements (see text). 4820 A. Mo ¨ glich et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Fig. 5. Dihedral angle analysis of HPr(WT) and HPr(I14A). Structural differences between the wild -type and mutan t form of HPr are visualized by a comparison of t he co rrespondin g backbo ne dihedral angles. Values for the wild-type and the mutant protein are indicated by w hite and g rey bars, respectively. The corresponding standard deviations are indicated by error bars. Significant variations between the two proteins are marked by black dots and indicate residues for which the absolute value of the difference in dihedral angles exceeds the sum of the errors. Ó FEBS 2004 Solution structure of the I14A mutant of HPr (Eur. J. Biochem. 271) 4821 Our data provide the experimental evidence of this hypothesis. After removing the hydrophobic isoleucine side chain by mutating residue Ile14 to a lanine, the conforma- tion of loop L1 is strongly changed due to a kink in helix a (Figs 4 and 5). Particularly the relative position of the catalytically active histidine i s clearly different an d less accessible to the solvent c ompared to the wild-type protein. These structural changes should also have a profound effect on the biological activity of H Pr(I14A). In agreement with this assumption we have found a much reduced phospho- transferase activity of the mutant compared to the wild-type protein in the standard complementation a ssay [48]. Reversal of the mutation-induced changes by xenon-binding One might reasonably assume that the removal of the bulky sidechain of an isoleucine residue via mutation to alanine simply leads to t he creation of a h ydrophobic c avity of corresponding size and shape. Our structural studies clearly show that this is not the case f or the mutant HPr(I14A). Although the general fold of t he protein is conserved, the overall conformation is changed leading to distinctly different structures for wild-type a nd mutant HPr with a RMSD of 0.155 nm for the backbone atoms (C a ,C¢, N). The replacement of t he large hydrophobic side chain of isoleucine with the much smaller one of alanine causes a collapse of the protein in that region. The resulting hydrophobic cavity is partly filled by side chain s of other hydrophobic residues. Helix a bends towards the protein interior to partially fill the void left by the removal of the isoleucine. Moreover, Leu86 undergoes a pro- nounced rearrangement of its side chain which also protrudes into t he space o ccupied by I le14 in the wild- type HPr. These structural changes induce further distor- tions of the c onformation of the m utant protein. However, despite all these structural rearrangements the surface map of HPr(I14A) shows that a small hydropho- bic cav ity remains (Fig. 6). The existence of this cavity was recently confirmed by xenon-binding studies [43]. Xenon atoms are known to bind preferentially into hydrophobic pockets of proteins [49–51]. Further, the difference in volume between the sidechains of isoleucine and alanine closely corresponds to the volume of a xenon atom, which has a van d er Waals radius of 0.217 n m. Most of the larger xenon-induced changes i n chemical shift were observed near the site of the mutation, which could readily be accounted for by the existence of a hydrophobic cavity [43]. In contrast, it was hard to rationalize why large changes were also observed for the C-terminal residues and why throughout the whole protein the xenon-induced shift changes were considerably larger than in the wild-type. An explanation for these findings is provided by this study. The chemical shift changes induced by xenon binding to the hydrophobic cavity of HPr(I14A) are strongly correlated with the corresponding differences of chemical Fig. 6. Hydrophobic cavity of HPr(I14A). The solvent-accessible sur- face of the HPr(I14A) molecule is shown. Residues 14, 15, 19 and 86 are coloured as in Fig. 4. A cavity in the reg ion where the m utation has been introduced is marked by the arrows. The existence of this cavity was confirmed by xenon-binding studies. Fig. 7. Changes in chemical shift ca used by xenon-binding and the Ile14Ala mutation. The normalized changes of the combined chemical shifts Dd tot /<Dd tot > of the amide groups are plotted as a function of their position in the sequ ence. T he comb ined ch emical sh ift cha nges Dd tot have been calculated according to Eqn (1). <Dd tot > represents the average value of the corresponding chemical shift values and is indicated by the broken line. Chemical shift changes were determined in 1 H- 15 N-HSQC spectra for t he wild-type protein and the mutant protein both in the absence and the p resence of xenon [43]. Xenon- induced chemical shift changes in HPr(I14A) (blue); chemical shift changes induced by the mutation in the absence of xenon (red). 4822 A. Mo ¨ glich et al.(Eur. J. Biochem. 271) Ó FEBS 2004 shifts between wild-type and mutant protein (Fig. 7). As detailed above the removal of the hydrophob ic sidechain of isoleucine effects profound structural rearrangements in HPr(I14A). Apart from two regions in the direct vicinity of the mutation site, strong structural differences are also observed for the C-terminus, most notably for Leu86. Furthermore, the whole structure displays a subtly different conformation (Fig. 4). All of these structural distortions are closely reflected in the xenon-induced chemical sh ift chan- ges. Large shift changes are mainly observed in the same two regions close to the hydrophobic cavity introduced by the m utation and near the C-terminus. In the other regions of the mutant protein smaller xenon-induced chemical shift changes, which are still significantly larger than those for wild-type HPr, are seen and are indicative of global if yet small conformational changes. Taken together, these find- ings imply t hat xenon-binding leads to a reversal o f the structural changes caused by the mutation. By binding to the h ydrophobic cavity, xenon shifts the conformational equilibrium of HPr(I14A) towards species closer resembling the wild-type struct ure. The smaller size o f the chemical shift changes caused by xenon-binding compared to the mutation-induced effects could be due to two reasons. On the one hand, xenon atoms bound to the protein could rapidly exchange with the bulk water [52]. Saturation could not be obtained with the pres- sures possible in our experimental setup. Therefore the observed shifts represent an average of the bound and the free state w ith the chemical shift changes being scaled down accordingly. On the other hand, xenon-binding does not necessarily reverse the mutation-induced effects completely. Conclusion The work presented here further supports the idea that high- pressure NMR studies are generally suitable to identify residues important for the stability and the function of proteins. Pressure changes could be used t o shift the equilibrium between different protein conformations [53]. In this way, it is possible to populate species only present to a small extent at atmospheric pressure. NMR spectroscopy is a convenient technique to monitor such changes with atomic resolution. Both structurally flexible residues, which m ight mediate the interaction with different ligands, and residues that stabilize the protein can be identified by this method. It would be interesting to see the influence of the mutation upon the pressure response of HPr. Currently, work is in progress to address this question. 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