Solution structure of the active-centre mutant I14A of the Andreas Mo¨glich1,*, Brigitte Koch2, Wolfram Gronwald1, Wolfgang Hengstenberg2, Eike Brunner1 and Hans Robert Kalbitzer1 1 Inst
Trang 1Solution structure of the active-centre mutant I14A of the
Andreas Mo¨glich1,*, Brigitte Koch2, Wolfram Gronwald1, Wolfgang Hengstenberg2, Eike Brunner1
and Hans Robert Kalbitzer1
1
Institute of Biophysics and Physical Biochemistry, University of Regensburg, Germany;2SG Physiology of Microorganisms, Ruhr-University of Bochum, Germany
High-pressure NMR experiments performed on the
histi-dine-containing phosphocarrier protein (HPr) from
Sta-phylococcus carnosushave shown that residue Ile14, which is
located in the active-centre loop, exhibits a peculiarly small
pressure response In contrast, the rest of the loop shows
strong pressure effects as is expected for typical protein
interaction sites To elucidate the structural role of this
residue, the mutant protein HPr(I14A), in which Ile14 is
replaced by Ala, was produced and studied by solution
NMR spectroscopy On the basis of 1406 structural
restraints including 20 directly detected hydrogen bonds, 49
1HN-15N, and 251HN-1Haresidual dipolar couplings, a well
resolved three-dimensional structure could be determined
The overall 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 addition, 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 represents 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 process, 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 Nd1atom 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 via the PTS is energetically favorable From the
residue His15 of HPr, the phosphoryl group can also be transferred to transcription factors containing PTS regula-tion domains (PRDs) Depending on their phosphorylaregula-tion 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 structures 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 structures known so far
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-dependent phosphotransferasesystem; RDC, residual dipolarcoupling.
*Present address: Department of Biophysical Chemistry, Biozentrum,
University of Basel, Switzerland.
(Received 28 July 2004, revised 13 October 2004,
accepted 21 October 2004)
Trang 2and two long a-helices Kalbitzer et al [16] used
high-pressure1H and15N NMR 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, which mainly consists of the b-sheet,
showed only little or moderate variation, large
pressure-induced changes of chemical shifts were observed in the
active site region encompassing residues 12–18 In contrast to
its surrounding residues, Ile14 displayed only a small
pressure-induced change of chemical shift Kalbitzer et al
[16] suggested that this residue, 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 and ligands
To evaluate this hypothesis, a mutant form of HPr from
S carnosuswas 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 The plasmid was overexpressed in E coli
strain BL21 DE3 HPr(I14A) was isolated as described
previously [18] Uniformly15N and15N/13C 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 mMpotassium phosphate, 100 mMKCl, 0.1 mMEDTA,
1 mMNaN3, 1 lMpepstatin, 1 lMleupeptin, 0.1 lMbovine
pancreatic trypsin inhibitor and, as an internal reference,
0.1 mM 2,2-dimethyl-2-silapentanesulfonic acid The pH
was adjusted to 7.14 by addition of KOH The final protein
concentration was between 1.4 and 1.7 mM 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 spectrometers
with1H resonance frequencies of 500, 600 and 800 MHz,
respectively All measurements were carried out at a
temperature of 298 K Time-domain NMR data were
processed using the XWINNMR package (Bruker) Proton
chemical shifts were assigned on the basis of 2D TOCSY
and HCCH-TOCSY spectra measured at 500 and
600 MHz, respectively Nitrogen (15N) and carbon (13C)
resonances could be determined from HSQC, HNCA, HNCO and CBCA(CO)NH spectra recorded at 600 MHz Distance restraints were derived from homonuclear 2D NOESY spectra in1H2O and2H2O and from a13C resolved 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 programPERMOL(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 programRELAX[20] These calculated spectra were compared with the experimental data The1H chemical shifts were referenced relative to 2,2-dimethyl-2-silapentane-5-sulfonic acid 15N and 13C reso-nances were referenced indirectly [21] Spectral visualization and volume integration of NOE signals was carried out using the computer programAUREMOL[22]
Determination of dihedral angles and hydrogen bonds Three-bond coupling constants between HNand Hdatoms,
3JHN-Ha, were measured by MOCCA-SIAM experiments [23,24] The values of the coupling constants were determined using the procedure described by Titman and Keeler [25] Structural restraints for the main chain dihedral anglesF 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 dipolar couplings requires partial molecular alignment of the sample molecules, which was obtained by the addition of 7.5% (w/v) of the DIODPC/ CHAPSO lipid mixture to the sample solution RDCs [30–32], were measured for the1HN-15N amide bond using both conventional nondecoupled1H-15N-HSQC and IPAP-[1H-15N]-HSQC experiments [33] RDCs were determined 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 Szz¼ 0.000491, Syy¼)0.000313,
Sxx¼)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 the1HN-1Ha-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
Trang 3resonance 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 1HN-1Ha-RDCs
could be determined with sufficient accuracy
Structure calculation
Structures were determined by simulated annealing
employ-ing version 1.0 of the computer programCNS[38,39] A total
number of 1406 nonredundant 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 between1H atoms were
derived from NOE cross-peak intensities in two- and
three-dimensional spectra The standard simulated annealing
protocol supplied with CNS was modified to allow two
different classes of residual dipolar couplings to be used as
restraints Apart from this, all other parameters
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 programMOLMOL[42] by fitting the positions of
the backbone atoms Ca, C¢ and N Structural images were
prepared with the computer programMOLMOLand rendered
withPOVRAY(http://www.povray.org)
Database deposition
Chemical shift values for1H,13C and15N 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
Ddtot between two states a and b are calculated from the
amide proton shifts, dH, and the amide nitrogen shifts, dN, in
these two states according to Eqn (1):
Ddtot¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
w2ðdaH dbHÞ2þ ðdaN dbNÞ2
q
ð1Þ Here, w denotes a weight factor that accounts for the
different sensitivities of the chemical shifts 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 standard deviations of the
chemical shifts of the amide nitrogen and proton 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 1H, 13C and 15N resonance lines have been obtained from conventional homonuclear and hetero-nuclear NMR experiments A list of the restraints used in
Table 1 Structural statistics of HPr(I14A) NMR experiments were conducted at 298 K and pH 7.14 Structures were calculated with CNS
using the standard simulated annealing protocol 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
Short and intermediate distance (i, i + j; 1 £ j £ 4)
365
F dihedral angles from 3 J couplings (MOCCA-SIAM)
44 Hydrogen bonds from H(N)CO experiment 20
1 H N - 15 N RDC from IPAP/HSQC experiments
49
1 H N - 1 H a RDC from MOCCA-SIAM experiments
25
Quality factors for the residual dipolar couplings Q
1
HN-15N residual dipolar couplings 0.2880
1 H N - 1 H a´ RDC residual dipolar couplings 0.4089 Restraint violations in the 10 lowest-energy
RMSD values for the
Core region (residues 2–9, 16–27, 32–37, 40–43, 47–53, 59–84),
backbone atoms Ca, 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 Ca, C¢, N 0.084
Ramachandran plot (except glycine and proline residues) Incidence
Energies of the 10 selected structures after refinement in water E/kJÆmol)1
Trang 4the structure calculation of HPr(I14A) is given in Table 1.
Interatomic distance restraints have been derived from
homonuclear and13C-resolved NOESY spectra Structural
restraints for the backbone dihedral angleF were calculated
from three bond J-couplings between HN and Haatoms
measured in MOCCA-SIAM spectra [24] Twenty
hydro-gen bonds could be directly detected in H(N)CO
experi-ments [28,29]
Using nondecoupled1H-15N-HSQC and IPAP-[1H-15
N]-HSQC experiments [33], RDCs for the 1HN-15N 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 coupling is strongly dependent upon the secondary
structure (Fig 1) Residues in a-helices mainly display
positive 1HN-15N 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 of largest
partial molecular orientation, is arranged almost parallel to
the a-helices and the b-sheet of the protein As the1HN-15N
bond vectors in the a-helices are therefore almost parallel to
the z-axis, their size is mainly determined by the positive
eigenvalue Szzof the tensor [31] In contrast, the1HN-15N
bond vectors in the b-sheets are almost perpendicular to
the z-axis and, therefore are determined by the negative
eigenvalues Syy and Sxx 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 HN and Ha 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 16 restraints per amino acid residue (Table 1) An average structure was calculated 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 (Ca, C¢, N) located in the core region of the molecule comprising the 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 connecting 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 Dependence of1HN-15N residual dipolar couplings on secondary
structure The size of the1H N - 15 N residual dipolar couplings is strongly
correlated with secondary structure A prediction of secondary
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 the figure has been restricted to the region of
)15 to 15 Hz.
Fig 2 Three-dimensional structure of HPr(I14A) relative to molecular alignment tensor The 3D structure of HPr(I14A) is shown relative to the principal axis system of the molecular alignment tensor determined for the1HN-15N residual dipolar couplings The secondary 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.
Trang 5programPROCHECK-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-helix
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 wild-type protein on the basis 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 were observed However, the extended water refinement protocol led to a significant improvement of the general geometry The structural statistics and thePROCHECK-NMRanalysis 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 protein, helix b is significantly shorter in the mutant protein and distorted at its C-terminal end In the core region 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 (Ca, C¢, N) of 0.119 nm When all backbone atoms of the proteins are taken into account, this value increases to 0.155 nm
Significant deviations between the two proteins are seen
in the active site region where the mutation has been introduced The replacement of Ile14 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.
The radius of the spline reflects the RMSD values of the Caatom
positions The scale bar indicates a length of 0.2 nm corresponding to a
RMSD value of 0.1 nm Residues are colour-coded according to the
number of restraints used in the structure 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
RMSD values for the
Core region (residues 2–9, 16–27, 32–37, 40–43, 47–53, 59–84), backbone atoms
Ca, 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 Ca, C¢, N 0.088
Ramachandran plot (except glycine and proline residues) Incidence (%)
Energies of the 10 selected structures
E total ) 11752 ± 456
Trang 6the isoleucine residue is instead partly filled by the backbone
and side 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 protein 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 direction 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 anglesF and Y of mutant and wild-type
protein also supports 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 found In addition, 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 is
created by the replacement of the bulky Ile14 by an
alanine (Fig 6) Potentially, the binding of xenon inside
this cavity could lead to a reversal of the structural
changes induced by the mutation because the size of an
isoleucine side chain almost exactly corresponds to that of
a xenon atom As1HNand15N chemical shifts provide a sensitive measure for the local structural environment of the amide bond, 1HN-15N-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 shift changes observed in the I14A mutant upon xenon-binding (Fig 7) While on average the total changes in chemical shifts due to the introduction of the mutation are about four times as large as those induced 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, nonlinear 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) The side chains of 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 chain in the mutant protein leads to significant structural rearrangements (see text).
Trang 7Fig 5 Dihedral angle analysis of HPr(WT) and HPr(I14A) Structural differences between the wild-type and mutant form of HPr are visualized by
a comparison of the corresponding backbone dihedral angles Values for the wild-type and the mutant protein are indicated by white and grey 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.
Trang 8Our data provide the experimental evidence of this
hypothesis After removing the hydrophobic isoleucine side
chain by mutating residue Ile14 to alanine, 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 is clearly different and less
accessible to the solvent compared to the wild-type protein
These structural changes should also have a profound effect
on the biological activity of HPr(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 assay [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 the creation of a hydrophobic cavity of
corresponding size and shape Our structural studies
clearly show that this is not the case for the mutant
HPr(I14A) Although the general fold of the protein is
conserved, the overall conformation is changed leading to
distinctly different structures for wild-type and mutant
HPr with a RMSD of 0.155 nm for the backbone atoms
(Ca, C¢, N) The replacement of the 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 chains
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 the space occupied by Ile14 in the wild-type HPr These structural changes induce further distor-tions of the conformation of the mutant protein However, despite all these structural rearrangements the surface map of HPr(I14A) shows that a small hydropho-bic cavity 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 der Waals radius of 0.217 nm Most of the larger xenon-induced changes in 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 region where the mutation 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 caused 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 sequence The combined chemical shift changes
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
in1H-15N-HSQC spectra for the wild-type protein and the mutant protein both in the absence and the presence 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).
Trang 9shifts between wild-type and mutant protein (Fig 7) As
detailed above the removal of the hydrophobic 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 shift
chan-ges Large shift changes are mainly observed in the same
two regions close to the hydrophobic cavity introduced by
the mutation 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 that xenon-binding leads to a reversal of the
structural changes caused by the mutation By binding to
the hydrophobic cavity, xenon shifts the conformational
equilibrium of HPr(I14A) towards species closer resembling
the wild-type structure
The smaller size of 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 with 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 to 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 might
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 The data also show that
with its affinity to hydrophobic cavities xenon can influence
conformational equilibria and thus can possibly restore
function by stabilizing the active conformation of a protein
Acknowledgements
The authors thank Dr Michael Wenzler, Dr Rolf Do¨ker, Jochen
Trenner and Dr Bernhard Ganslmeier for helpful discussions, and
Christian Gro¨ger for recording 1H-15N-HSQC spectra Financial
support by the Deutsche Forschungsgemeinschaft (Br 1278/9–1, SFB
521 projects A6, C6), the Fonds der chemischen Industrie and the EU
(FP6, SPINE-consortium) is gratefully acknowledged Thanks are
further due to Ms Ingrid Cuno for carefully proofreading the
manuscript.
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