The homogeneity of the values of the overall correlation time calculated from the individual R2⁄ R1 ratios suggested a good degree of isotropy of the global molecular motion, consis-tent
Trang 1transcription factor 1 homeodomain – hints from 15N-NMR relaxation studies
Devrim Gu¨mral, Luana Nadalin, Alessandra Corazza, Federico Fogolari, Giuseppe Damante,
Paolo Viglino and Gennaro Esposito
Dipartimento di Scienze e Tecnologie Biomediche, Universita` di Udine, Italy
Homeodomains (HDs) comprise a very well-known
class of DNA-binding domains occurring in a large
family of transcription activators involved in the
determination of cell development [1–3] The tertiary
structure of the HD of rat thyroid transcription
fac-tor 1 (TTF-1), a 67-residue domain, was determined
by NMR spectroscopy [4] (Brookhaven Protein Data
Bank ID code 1FTT) The whole TTF-1 protein
(378 residues) is responsible for transcriptional
activa-tion of genes expressed only in follicular thyroid cells [5] and lung epithelial cells [6] The structural fea-tures of the TTF-1 HD are the typical ones observed
in HDs, i.e three helices (Gln10–Gln22, Ala28–Ile38, Thr43–Gln59) connected by a loose loop (Gln23– Ser27) between helix I and helix II and by a tight turn (His39–Pro42) between helix II and helix III (helix–turn–helix motif; Fig 1) The DNA recogni-tion helix (helix III) is fairly ordered also in the
Keywords
backbone dynamics; model-free approach;
NMR15N relaxation; spectral density
mapping; thyroid transcription factor 1
homeodomain
Correspondence
G Esposito, Dipartimento di Scienze e
Tecnologie Biomediche, Universita` di Udine,
P.le Kolbe, 4, 33100 Udine, Italy
Fax: +39 0432494301
Tel: +39 0432494321
E-mail: gesposito@mail.dstb.uniud.it
(Received 20 October 2007, revised 25
November 2007, accepted 28 November
2007)
doi:10.1111/j.1742-4658.2007.06208.x
The backbone dynamics of the 15N-labeled homeodomain of the rat thy-roid transcription factor 1 has been studied by 2D NMR spectroscopy Longitudinal (R1) and transverse (R2) 15N relaxation rate constants and steady-state {1H}–15N NOEs were measured at 11.7 T These data were analyzed by both the model-free formalism and the reduced spectral den-sity mapping (RSDM) approaches The global rotational correlation time,
sm, of the thyroid transcription factor 1 homeodomain in aqueous solution
at 286 K was found to be 10.51 ± 0.05 ns by model-free formalism and 9.85 ± 1.79 ns by RSDM calculation The homogeneity of the values of the overall correlation time calculated from the individual (R2⁄ R1) ratios suggested a good degree of isotropy of the global molecular motion, consis-tent with the similar global smresults obtained with the two different meth-ods Tyr25 was found to undergo slow conformational exchange by both methods, whereas this contribution was identified also for Lys21, Gln22, Ile38 and His52 only by RSDM With both methods, the C-terminal frag-ment of helix III was found to be more flexible than the preceding N-termi-nal portion, with slightly different limits between rigid and mobile moieties Additionally, Arg53 appeared to be characterized by an intermediate motional freedom between the very flexible N-terminal and C-terminal resi-dues and the structured core of the molecule, suggesting the occurrence of
a hinge point Finally, slow-time-scale motions observed at the end of helix I, at the end of helix II and within helix III appear to be consistent with typical fraying transitions at helical C-termini
Abbreviations
Antp, Antennapedia; HD, homeodomain; MD, molecular dynamics; MF, model-free; RSDM, reduced spectral density mapping; TTF-1, thyroid transcription factor 1.
Trang 2absence of DNA, as first reported for Antennapedia
(Antp) HD [7] For the TTF-1 HD, a discontinuity
of the hydrogen bond network between N-terminal
and C-terminal moieties of the recognition helix was
observed at the highly conserved fragment Asn51–
His52–Arg53 [4], suggesting the occurrence of either
a kinking or tightening of the local geometry A
similar discontinuity had been noted in solution also
in the Antp HD [8,9] and the Antp (C39S) HD [10],
and indeed, originally, the C-terminal extension of
helix III, i.e residues 53–59, was proposed to form
helix IV However, in the absence of direct evidence
supporting a structural interruption of the geometry
of the recognition helix for either Antp or the TTF-1
HD, the anomalous amide exchange pattern and the
NOE connectivity data of the C-terminal portion of
helix III had to be ascribed to local mobility effects
[4,10] Subsequently, a quantitative analysis of
1H–2H exchange rates of the TTF-1 HD revealed
opposite effects to helix III stability within the
frag-ment 51–53 that may be relevant to the
conforma-tional dynamics of the HD recognition helix upon
DNA binding [11]
In the following, we present a 15N-NMR relaxation study of the rat TTF-1 HD to address the backbone dynamics in solution 15N-NMR as well as 13C-NMR relaxation studies can be usefully applied to determine the dynamics of proteins [12,13] In high magnetic fields, the relaxation of these nuclei is mainly governed
by dipole–dipole and chemical shift anisotropy mecha-nisms For globular proteins, the analysis of the exper-imental relaxation data by means of the model-free (MF) approach [14,15] provides a description of the motions in terms of global overall rotational correla-tion time, sm, a generalized order parameter, S2, and
an effective internal correlation time, se For15N relax-ation data, the generalized order parameter reflects the amplitude of the internal motion of the15N–1H vectors
in the fast ps-to-ns time range An alternative method established to examine 15N–1H vector mobility is based
on the estimation and interpretation of the spectral density values from the individual relaxation rates [16–22], an approach most commonly applied in a restricted version referred to as reduced spectral den-sity mapping (RSDM) This method provides an anal-ysis of protein dynamics that requires no model assumptions It gives spectral density values at J(0), J(xN) and J(<xH>), directly calculated from the measured relaxation parameters, that contain contribu-tions from the overall as well as the local dynamics Graphical analysis of the spectral density values pro-vides a qualitative picture of the internal motions with
no bias, as the whole approach does not make any assumption about the motions to be investigated
Results
Relaxation parameters The individual R1, R2 and NOE values of the back-bone amide 15N nuclei of the TTF-1 HD at 286 K are given in supplementary Table S1, Table S2 and Fig S1 Side-chain nitrogens were not considered for analysis, except for the indole nitrogen of Trp48, which represents a convenient probe with which to monitor the dynamics of the HD hydrophobic core (supplementary Table S1)
The longitudinal relaxation rates range between 1.15 and 1.97 s)1 The lowest R1 values are observed for Lys24 and Met37 and the residues of the flexible termi-nal segments, with a characteristic pattern of decreas-ing values on approachdecreas-ing these latter segments from the respective adjacent helices The highest R1 values are observed for Ser27, Arg31, Glu32, Ser36, Ile38, Val45, and Trp48 The transverse relaxation rate val-ues, higher than the corresponding R1 constants by
Fig 1 Cartoon of the TTF-1 HD backbone (Protein Data Bank code
1FTT) [4] with helix I (brown), helix II (magenta), and the DNA
rec-ognition helix, helix III (green) The side chains of the residues
whose 15 N– 1 H vectors undergo slow motions, i.e ls-to-ms (Lys21
and Gln22 in helix I, Tyr25 in the large loop, Ile38 in helix II, and
His52 in helix III), are in blue, whereas, for Leu26, the red color
indicates the coupling of low-frequency and high-frequency
dynam-ics With the exception of His52, all the mentioned residues are
located in the hydrophobic core of the molecule (i.e Ile38) or close
to residues of this core (i.e Lys21 and Gln22, neighboring Phe20;
Tyr25, neighboring Leu26) The drawing was prepared using
Open-Source PYMOL (DeLano Scientific LLC, South San Francisco, CA,
USA).
Trang 3one order of magnitude, fall in the range 8.41–
16.53 s)1 The lowest R2 values are shown by the
N-terminal and C-terminal residues and by Leu34,
Gln44, Arg53, Arg58 and Gln59 A unique value of
26.39 s)1, by far the highest one, is observed for
Tyr25, which strongly suggests the presence of a local,
low-frequency conformational exchange contribution
The steady-state {1H}–15N NOEs span the interval
)1.70 ⁄ +0.89 Negative values are observed for the
ter-minal fragments, i.e Arg1–Leu7 and Lys61–Gln67,
reflecting the local dynamics characterized by fast
motions In particular, the sign inversion transitions of
NOEs, seen on approaching the helical tracts from
flexible terminal residues, parallel the similar trends
observed for relaxation rates, and reflect consistently
the changes in local motional properties In the
recog-nition helix, lower NOE values are obtained for the
C-terminal moiety, confirming that it is more flexible
than the N-terminal one The highest {1H}–15N NOEs
were measured for Glu17 in helix I, Ser27 and Leu34
in helix II, and Lys46 and Gln50 in the N-terminal
portion of helix III For an isotropically tumbling
globular molecule, in the absence of internal motions
and with relaxation due to dipole–dipole and chemical
shift anisotropy mechanisms, {1H}–15N NOEs can be
shown to span values between )3.6, for xNsm<<1,
and +0.82, for xNsm>>1, where smis the global
over-all rotational correlation time [13] Within the
esti-mated uncertainty, the residues that show a {1H}–15N
NOE higher than the theoretically estimated maximum
are Glu17, Leu34 and Lys46 This is conceivably a
consequence of the overlap affecting the corresponding
resonance Therefore, the experimental data of these
three residues were not further considered for
subse-quent MF and RSDM analysis calculations However,
the qualitative implication of a high {1H}–15N NOE
for Glu17, Leu34 and Lys46, i.e low specific mobility,
is consistent with the NOE trend of the corresponding
adjacent residues and hence does not conflict with the
global interpretation of the data With the exclusion of
the N-terminal octapeptidyl and C-terminal
nonapept-idyl fragments of Glu17, Leu34 and Lys46, the average
of the {1H}–15N NOEs is 0.68 ± 0.10 (supplementary
Table S2) This value can be reliably considered to be
the average NOE over the structured core of the
inves-tigated TTF-1 HD molecule
MF motional parameters
Figure 2 shows the individual overall rotational
corre-lation time, smi, calculated from the individual residue
R2⁄ R1ratios, the generalized order parameters, S2, and
the effective correlation times, se, of the TFF-1 HD
from MF analysis of the 15N relaxation parameters at 11.7 T and 286 K with the corresponding uncertainties (the actual values are listed in supplementary Table S3) No other exchanging contributions, Rex, but
Fig 2 Bar graphs of overall rotational correlation time, s mi (ns), generalized order parameter, S 2 and effective internal correlation time, se(ps) values as a function of the TTF-1 HD sequence The parameters were obtained from measurements at 11.7 T and
286 K seand S 2 values were not calculated for Glu17, Leu34 and Lys46, as their NOE signals exhibited almost 100% overlap Addi-tional blank slots in the correspondence of residues 29 and 42 are for prolines The s e values of Ser27 and Gln50 are not reported, because they were not optimized by MF analysis The extension of TTF-1 HD helical segments is depicted above the graphs.
Trang 4that expected for Tyr25 (14.67 ± 2.35 ns) were found
from MF formalism calculations
Rotational correlation time
From the estimates of smi based on the individual
relaxation rate ratios (Fig 2), an average value of
9.7 ± 0.4 ns is extracted for the overall tumbling by
considering only the parameters from the best defined
(and conceivably most rigid) regions of the TTF-1 HD
(Gln10–Gln22, Ala28–Ile38, Thr43–Gln50) as
deter-mined from the NMR structure of the molecule [4]
When averaging is extended over the whole smi
dataset, only a slight difference is obtained, i.e
Æsmiæ = 9.5 ± 0.9 ns The excellent agreement between
the averages shows that the local segmental mobility
differences, albeit remarkable as inferred from the
NOE data, have little effect on the value of the Æsmiæ
estimate, and adds confidence to the assumption of
isotropic motion adopted by the equation of the
relax-ation rate ratio [13] All the individual smi values lie
within 2r from average (95% confidence level), except
for Tyr25, due to the high value of the corresponding
R2constant, which is affected by a slow exchange
con-tribution A more accurate estimate of the global sm,
obtained by unbiased grid search optimization over the
experimental parameters and subsequent Brent
minimi-zation [23], in the context of MF calculations, gave a
value of 10.51 ± 0.05 ns, i.e slightly higher but not
far from the value computed from relaxation rate
ratios
Local generalized order parameters and internal
effective correlation times
Besides the optimization of the molecular tumbling
rate, MF analysis of relaxation data provides a set of
optimized parameters describing local motions Except
for Tyr25, all the 15N relaxation data of the TTF-1
HD were satisfactorily fitted by means of a
dual-motion model entailing a single-frequency local
fluctu-ation superimposed on the global motion The quality
of the fitting was statistically validated by v2test
against the corresponding parameter distribution of
Monte Carlo simulations The individual generalized
order parameters and internal effective correlation
times are plotted in Fig 2 Their values reflect,
respec-tively, the specific amplitude and the frequency of the
local fluctuations for the motion of each considered
in-ternuclear 15N–1H vector The lowest S2 values and,
correspondingly, the shortest se values are obtained
at the N-terminal and C-terminal fragments 1–7 and
60–67 of the TTF-1 HD This pattern suggests wide
motional freedom of the 15N–1H vectors, which is in line with the disordered NMR structure observed for the same regions [4] The N-terminal flexibility starts
to quench before reaching helix I, at Phe8 and Ser9, where both parameters of local backbone dynamics increase This progressive transition pattern is attrib-uted to the involvement of Ser9 in the N-capping motif
of helix I [4] The trend of the effective internal corre-lation time, se (referred to as local correlation time), along helix I features a behavior that appears typical within the whole set of MF-based parameters obtained for the TTF-1 HD, namely an increase of local corre-lation time with increasing generalized order parame-ter This behavior is intriguing when compared to the established expectation that associates limited local motional amplitudes, i.e S2 between 0.8 and 1, with fast local motions, i.e small se, and, conversely, wide local motional amplitude, i.e S2< 0.8, with slow local motion, i.e large se In other words, most often for the TTF-1 HD, S2 and seexhibit an opposite correla-tion from what is expected This casts substantial doubts on the reliability of the picture emerging from the application of MF formalism to TTF-1 HD relaxa-tion data In detail, the highest S2 values are obtained for Gln50 and Tyr54, two residues that are essential for the DNA recognition specificity of the TTF-1 HD [24,25] The restriction in local motion amplitude, implied by the values of S2, seems consistent with the role of Gln50 and Tyr54, but the corresponding se val-ues are not easily rationalized For Gln50, the optimi-zation procedure fails to fit the experimental data with
se£ 11 000 ps A low frequency of the internal motions could be considered to match the above-men-tioned correlation between high S2 and large sevalues
In contrast, for Tyr54 a very low value of the opti-mized se (296 ± 192 ps) is obtained, which is difficult
to reconcile with the pattern most commonly observed
in the dataset, when S2 is close to 1 The physical picture for Tyr54 becomes consistent with local fluctu-ations with remarkably limited amplitude and high fre-quency The high level of uncertainty affecting se of Tyr54 may suggest that the result should be considered
as a numeric artefact of the optimization However, a decreasing trend of the internal secoupled with a simi-lar behavior of the generalized order parameter unequivocally emerges on examination of seg-ment 50–55 of the TTF-1 HD (Fig 2) Besides Gln50, optimization fails to retrieve a se£ 11 000 ps also for the data of Ser27, a residue of the loop between helix I and helix II In this case, however, the generalized order parameter is, within the estimated error, lower (0.755 ± 0.072) than the average value observed in the structured molecular core (0.86–0.87) Although the
Trang 5large error in S2 may reflect some problems with the
available data quality, a reduced motional rate for the
Ser27 backbone appears to be plausible, considering its
involvement in the defective capping of helix II [4] At
this stage, the results are better described by
consider-ing the average values observed in the different
second-ary structure elements as reported in Table 1
The local dynamics of the three helical regions of
the TTF-1 HD look very similar when only the
aver-age generalized order parameters are considered A
clear difference emerges, however, if the internal
corre-lation times are taken into account Only the motion
of helix I appears quite uniform, as inferred from the
similar values of the mean and weighed mean se
Helix II shows the largest variability in local
fluctua-tion frequency, despite the fact that the relative mean
generalized order parameter and the standard
devia-tion are very close to the corresponding counterparts
of helix I This result can be rationalized on a
struc-tural basis Helix II, in fact, should be the least stable
among the TTF-1 HD helices, because of its
incom-plete hydrogen bond network, which is due to
defec-tive N-capping and distortions introduced by Pro29
At the same time, the side chains of residues 34, 35
and 38 are tightly anchored in the hydrophobic core of
the molecule, whereas the Glu30 side chain is involved
in a salt bridge [4] The restricted mobility of four side
chains, out of 10 in helix II, appears to be coupled to
a lower motional frequency of the corresponding
or adjacent backbone amide bond vectors, which
accounts for the inhomogeneity of the local correlation
times For helix III, the inhomogeneity can be easily
appreciated by inspecting Fig 2, where the well-known
difference between the N-terminal and C-terminal
moieties of the recognition helix can be seen If the
MF parameter averages of Table 1 are accordingly split into average values for segments 43–52 and 53–
59, some internal motion inhomogeneity of helix III is seen to occur also within the single fragments The N-terminal portion exhibits slightly higher <S2> and standard deviation than helix I and helix II, and a broad distribution of se, with a weighted average around 1.5 ns, like helix II Again as with helix II, some side chains in this part of the recognition helix (residues 45, 48 and 49) contribute to the hydrophobic core of the molecule Thus, hydrophobic core anchor-ing has similar results for internal fluctuations in helix II and the N-terminal moiety of helix III
Overall, it seems that the whole motional regime of the TTF-1 HD, in the experimental conditions chosen for obtaining the relaxation data (286 K), matches only poorly (and qualitatively) the behavior needed to comply with the implicit conditions imposed by the
MF approach In most cases, an increase⁄ decrease in the generalized order parameter corresponds to an increased⁄ decreased se, which calls for a motional regime that appears to be inconsistent within the MF framework However, all attempts to fit the experimen-tal data with the extended MF approach [26], which uses a double-timescale model for internal motions, were also unsuccessful It is tempting to speculate that the physically puzzling picture emerging from the MF-based fitting of the majority of the TTF-1 HD relaxa-tion data could be attributed to correlated local dynamics that occur on a timescale similar to that of the overall tumbling
Graphical analysis of spectral densities Spectral densities at three frequencies [J(0), J(xN) and J(0.87xH)] were calculated according to the matrix equation given in supplementary Doc S1 The individ-ual spectral density values along the sequence of the TTF-1 HD are displayed in the bar graphs of Fig 3, and the corresponding numerical values are given in supplementary Table S4 Linear correlations between J(0) and J(xN) and between J(0) and J(0.87xH) for the TTF-1 HD were then examined as proposed by Lefe`-vre et al [21] The fit was obtained by linear regression, and only the corresponding J(0)–J(xN) correlation plot is shown in Fig 4
The localization of the experimental points in Fig 4 along the correlation line is directly related to the dis-tribution of the energy between the overall tumbling and the internal mobility, and is indicative of the degree of internal restraint of each 15N–1H vector motion In Fig 4, most of the points cluster in the same region The dashed curve, called the theoretical
Table 1 Mean values and corresponding standard deviations (in
parentheses) of S2(dimensionless) and s e (ps) parameters for the
secondary structure elements of the TTF-1 HD at 286 K.
Structural unit ÆS 2 æ Æs e æ Æs e æ wa
Helix I (10–22) 0.87 (0.04) 1983 (406) 1964 (37)
Helix II (28–38) 0.86 (0.04) 2493 (1374) 1561 (57)
Helix III (43–59) 0.85 (0.08) 1486 (780) 1008 (19)
Helix III (43–52) 0.87 (0.06) 1885 (710) 1596 (51)
Helix III (53–59) 0.82 (0.09) 1030 (613) 905 (21)
Helix III (42–56) 0.87 (0.06) 1630 (809) 1297 (37)
Helix III (51–56) 0.86 (0.06) 1345 (740) 1142 (47)
Loop (23–27) 0.84 (0.08) 1038 (542) 468 (47)
Tight turn (39–42) 0.92 (0.04) 1960 (1047) 1403 (106)
N-terminus (1–9) 0.63 (0.19) 805 (752) 276 (3)
C-terminus (60–67) 0.58 (0.09) 514 (277) 206 (3)
a
Weighted average calculated using the individual s e uncertainties
(ri) as weighting factors (1 ⁄ r i2).
Trang 6curve, indicates the spectral density values expected for
a simple Lorentzian model of J(x) calculated over a
very wide range of correlation times, s Most of the
experimental points accumulate rather close to the upper intercept of the theoretical curve and the fitting (solid) line, where the motion of a unique15N–1H vec-tor is defined by a single Lorentzian function with a global overall rotational correlation time, sm The points from N-terminal and C-terminal residues (Arg1–Leu7 and Ala60–Gln67), together with those from Arg58 and Gln59 in the C-terminal end of helix III, are located apart from the major cluster, towards the lower intercept of the theoretical curve and fitting line (Fig 4, left inset), where the motion of
Fig 3 Bar graphs of spectral density function values (ns) at the
zero, x N and 0.87x H frequencies, versus the sequence of the
TTF-1 HD Measurements were done at 11.7 T and 286 K Blank
slots are for residues 29 and 42 (prolines) and Glu17, Leu34 and
Lys46, which were excluded because of the extensive overlap
affecting the corresponding signals Correlations were calculated by
means of MATHEMATICA 5.2 software, using the relaxation dataset
given in supplementary Table S2 Relaxation data obtained from
lin-ear prediction were used for calculation only when the error
intro-duced by the procedure was acceptable, as discussed in
supplementary Doc S1 The extension of TTF-1 HD helical
seg-ments is depicted above the graphs.
Fig 4 J(xN)–J(0) correlation for the individual residues of the
TTF-1 HD from15N relaxation measurements Different colors are used
to indicate the distinct groups of residues along the sequence, i.e N-terminal (orange), C-terminal (violet), helix I (yellow), helix II (pink), helix III (green), loop (cyan), tight turn (brown), and residues that undergo conformational exchange motions (blue) The fit (dark solid line) was obtained by linear regression with the exclusion of Arg1 and Gln67 (which exhibit strong negative NOE values) and Lys21, Gln22, Tyr25, Ile38 and His52 [which make conformational exchange contributions to J(0)] The dashed curve (theoretical curve) was calculated for J(0) and J(x N ) as a function of s , using a simple Lorentzian function The left-hand inset shows an overview
of the theoretical curve and the fitting line to highlight the two intercept points The right-hand inset shows Tyr25 correlation, which occurs outside the plotted area Analytically, J(0.87x H ) depends only on the cross-relaxation rate; that is, it is largely deter-mined by the heteronuclear NOE and thus it is most sensitive to high-frequency motions of the protein backbone On the other hand, the value of J(xN) is extracted also from R1, whereas J(0) is determined also by both R1and R2 Therefore, J(0) is sensitive to both nanosecond timescale motions and contributions from
ls-to-ms slow exchange processes For this reason, the main informa-tion on dynamics can be derived from analysis of J(0) A plot of the correlation J(0.87x H )–J(0) is given in supplementary Fig S2.
Trang 7a unique15N–1H vector is defined by a single
Lorentz-ian function with a fast s that is interpreted as
general-ized internal correlation time, sgi For any point
between the upper and lower intercepts of the
theoreti-cal curve with the fitting line, the spectral density
func-tion can be expressed as a linear combinafunc-tion of the
two Lorentzian functions defined by smand sgi,
respec-tively The proximity to one of the intercepts between
the theoretical and fitting curves reflects the relative
contribution of each component Lorentzian function
to the specific spectral density of each experimental
point Therefore, according to the RSDM analysis
[21], most of the 15N–1H vectors of the TTF-1 HD
core move at the rate of the overall rotational
correla-tion frequency, and relaxacorrela-tion mainly occurs as a
result of overall rotational diffusion Among all the
TTF-1 HD backbone 15N–1H vectors, those from
dis-ordered N-terminal and C-terminal residues, together
with Arg58 and Gln59, are the most mobile ones and
exhibit fast-timescale (ps-to-ns) motion
In Fig 4, the points corresponding to residues
Lys21, Gln22, Tyr25, Ile38 and His52 are shifted to
the right above the theoretical line, which is a
typi-cal pattern for the occurrence of a slow (ls-to-ms)
exchange process The data relative to Lys24 and
Met37, together with those of several terminal
resi-dues (Arg1, Arg2, Ala64, Gln66 and Gln67), fall
outside the major cluster of points and feature a
dis-tinct dynamic behavior as compared to the
remain-ing 15N–1H vectors of the core Their spectral
density functions cannot be expressed with only two
Lorentzian functions
In the tightening⁄ kink of the recognition helix
introduced by the Asn51–His52–Arg53 tripeptide,
His52 and Arg53 show rather different dynamic
behaviors Arg53 appears to possess an intermediate
motional freedom between those of the N-terminal
and C-terminal residues and the core; that is, it
undergoes ps-to-ns timescale motion On the other
hand, His52 shows slow conformational exchange
contributions in the ls-to-ms timescale, as mentioned
above A similar situation is observed for the pairs
Glu30–Arg31 and Gln44–Val45, with the first residues
exibiting faster motions (ps-to-ns timescale), and the
latter residues slower motions on the nanosecond
timescale
Detailed analysis of the spectral density functions
can be performed using the bar charts of Fig 3 to
obtain the individual dynamic properties of each
15N–1H vector It can be seen that the15N–1H vectors
of the N-terminal and C-terminal residues undergo the
most rapid motions as compared to the rest of the
TTF-1 HD backbone This is highlighted by low J(0)
and J(xN) values and correspondingly high J(0.87xH) values, a pattern that is typically expected when the considered internuclear vectors reorient on a fast (ps-to-ns) timescale
In the loop between helix I and helix II, Tyr25 shows a J(0) value that is much higher than that of any other15N–1H vector of the backbone This pattern suggests that a slow exchange process in the ls-to-ms range occurs at Tyr25, because such processes increase the value of the spectral density function in the low-frequency range, i.e from zero to a few kilohertz, but have no influence at high frequencies, i.e in the mega-hertz range On the other hand, the significant J(0.87xH) value of Tyr25 indicates mobility Within the residue group with increased J(0), however, Tyr25 N–H and, to a lesser extent, His52 N–H appear
to undergo some additional fast motions, as shown by higher J(0.87xH) values
In general, flexibility is observed at the loop resi-dues, but not in the tight turn For helix I and helix II, J(0) values show a quite regular distribution along the sequence if residues undergoing exchange are excluded (Fig 3) J(xN) and J(0.87xH) values are fairly constant along helix I [except for the higher value of J(0.87xH) for Leu16] and are more dispersed along helix II For the latter, this indicates segmental mobility being adopted with a less defined secondary sturucture, prob-ably resulting from the lack of a complete hydrogen bond network [4] The dynamics of helix III can be divided into two different regions, with a border occur-ing at His52–Arg53 for all spectral density values The C-terminal segment of helix III (Arg53–Gln59) has lower J(0) values than the adjacent N-terminal moiety and the whole core of the TTF-1 HD, reflecting mobil-ity related to poorly defined secondary structure [4] Conversely, at the N-terminal segment of helix III, higher J(0) values are inferred from analysis, consistent with the better defined and more stable secondary structure
Overall, apart from the singularity at His52 that results from an exchange contribution due to slow aro-matic ring motion, as previously described, J(0) values are seen to vary along helix III with some regularity within the two identified moieties, i.e a slight decrease along segment 47–53, followed by a slight increase
in segment 53–55 The J(0) minimum is reached at Arg53, where the low-frequency motion profile shows similar characteristics as found at Arg58 for Gln59, the frayed extremity of the recognition helix The pat-tern described for J(0) is observed also for J(xN) along helix III, again with a minimum at Arg53 In a quite complementary fashion, J(0.87xH) reaches a maximum
at Arg53, with a subsequent abrupt decrease at Tyr54
Trang 8[in correspondence with the increases in J(0) and
J(xN)] and then a progressive increase on moving
towards the end of the recognition helix The whole
picture outlines the peculiar dynamic profile of a hinge
point at Arg53 that exhibits conspicuous minima
of J(0) and J(xN) and a significant maximum of
J(0.87xH), and that emerges not only within helix III,
but also over a large portion of the protein, from Phe8
to Met56, including the loop and the tight turn At the
same time, in the vicinity of the Arg53 hinge point,
precisely at Glu50, a minimum of J(0.87xH) occurs,
along with correspondingly high values of J(0) and
J(xN), an indication of slow local motion consistent
with the presence of a hydrogen bond network that
restricts the excursion of the Glu50 backbone Other
relevant details of the spectral density analysis are seen
for Lys24 and Met37 amides, where increased values
of J(0) are coupled to low J(xN) values Although
significantly low values of J(xN) are considered to be
evidence for fast motions, the corresponding J(0.87xH)
of the same residues rather suggests more complex
dynamics, i.e other than the dual low-frequency and
high-frequency motional regime that appears to govern
local dynamics elsewhere, e.g Leu26
Table 2 lists the mean J(x) values together with
the corresponding standard deviations for the
differ-ent secondary structure elemdiffer-ents of the TTF-1 HD
It is readily seen that the 15N–1H vectors of helix I,
helix II and the N-terminal segment of helix III do
not show major differences in the J(x) values
Con-versely, the C-terminal fragment of helix III has
lower mean values for both J(0) and J(xN), and a
significantly higher mean value for J(0.87xH), which
further stresses the different dynamic behaviors of
the N-terminal and C-terminal segments of the
rec-ognition helix
Global overall and generalized internal correlation times
The roots of the third-order polynomial proposed by Lefe`vre [21] were calculated for both linear correla-tions of J(xN) and J(0.87xH) versus J(0) (see supple-mentary Fig S2) to evaluate smand sgi From J(xN)– J(0) correlation, only two physically meaningful solutions were obtained, i.e sm= 9.85 ± 1.79 ns and
sgi= 0.28 ± 0.11 ns J(0.87xH)–J(0) correlation yielded three roots, one for sm (9.84 ± 0.20 ns) and two for sgi (0.26 ± 0.03 ns and 0.55 ± 0.06 ns) (sup-plementary Doc S1 and Fig S2)
Comparison of results from MF and RSDM The results for sm obtained by the MF and RSDM approaches are in fairly good agreement, especially if the comparison is drawn using the average value esti-mated from R2⁄ R1ratios Therefore, the assumption of isotropic overall rotational diffusion for the TTF-1
HD proves to be convincingly appropriate
The generalized order parameter values obtained from the MF approach are consistent with the results
of RSDM Lower generalized order parameters are obtained for N-terminal and C-terminal residues, for the loop, and partially for helix III, pointing to large-amplitude motions Higher generalized order parame-ters are obtained for the structured regions as well as the tight turn, indicating restricted mobility, in agree-ment with the RSDM results Most of the effective internal correlation times obtained by the MF approach appear to be unreliable within the framework
of the theory This could arise from the very well-known limitations of MF formalism for the case of internal motions occurring on a timescale similar to that of the overall tumbling [27] In this case, such slow motions would superimpose faster internal motions, leading to a situation that would not match the regime supporting the assumption of MF formalism This is also assumed to be the reason why we were not able to
fit our data using an extended MF formalism [26,27] Although anomalously high se values were often obtained, it is worth noting that MF calculations gave high S2and correspondingly relatively low sevalues for Lys24, Glu30, Gln44 and Tyr54, indicative of restricted amplitude and fast-timescale motions that are consis-tent with the corresponding results from RSDM Also, the relatively decreased S2 and the corresponding rela-tively low se values (sub-nanoseconds) for Arg53, Arg58 and Gln59 suggest less restrictive and faster local motions that are consistent with the reduced spectral density results In the ls-to-ms timescale, only Tyr25
Table 2 Mean spectral density values (ns) and corresponding
standard deviations (in parentheses) for the secondary structure
elements of the TTF-1 HD at 286 K.
Structural unit J(0) J(x N ) J(0.87x H )
Helix I (10–22) 3.78 (0.30) 0.354 (0.008) 0.008 (0.001)
Helix II (28–38) 3.84 (0.37) 0.355 (0.029) 0.008 (0.001)
Helix III (43–59) 3.63 (0.34) 0.343 (0.020) 0.011 (0.005)
Helix III (43–52) 3.83 (0.26) 0.354 (0.017) 0.008 (0.002)
Helix III (53–59) 3.38 (0.25) 0.329 (0.014) 0.014 (0.006)
Loop (23–27) 3.79 (0.12)a 0.340 (0.024) 0.010 (0.003)
Tight turn (39–42) 3.69 (0.11) 0.342 (0.010) 0.008 (0.001)
N-terminus (1–9) 2.83 (0.52) 0.271 (0.065) 0.027 (0.013)
C-terminus (60–67) 2.62 (0.30) 0.262 (0.052) 0.030 (0.006)
a Tyr25 was excluded to avoid a significant bias on the average
from the slow exchange contribution (see text).
Trang 9was found to have exchange contributions using MF
analysis, whereas by RSDM, Lys21, Gln22, Ile38 and
His52 were also identified
Molecular dynamics (MD) simulation results
Snapshots were taken at 500 ps intervals in order to
obtain a statistical ensemble for the system studied
The overall flexibility of the molecule was assessed by
the average rmsd of the backbone atoms when the core
region spanning residues Gln10–Arg58 was
superim-posed between all snapshot pairs From this analysis,
the conformational freedom of the N-terminal and
C-terminal regions was apparent, with average rmsd
values up to 10 A˚ The average rmsd values for the
rest of the molecule spanning residues Gln10–Arg58
were mostly < 1.0 A˚ The analysis of the correlation
function of the N–H vectors was more informative,
although less straightforward The short simulation
time precludes a direct spectral density analysis In
order to highlight local motions, the global rotational
motion of the molecule must first be assessed This
was done by superimposing the core of all snapshots,
taken at 100 ps intervals, on the snapshot with the
smaller average rmsd The correlation function C(i,m)
was defined as the average of the position vector scalar
product ~rNHðtÞ ~rNHðt þ mDtÞ over the trajectory for
residue i The root mean square of the quantity
[1) C(i,m)] was thus indicative of the deviation of the
vector N–H of residue i from the global behaviour
This procedure is solely motivated by the inadequate
time sampling provided by a 10 ns MD simulation
The largest deviations from global behavior are
observed at the N-terminus and C-terminus, with a
transition from disordered to more ordered vectors
between Phe8 and Ser9, and between Gln59 and
Arg58 Interestingly, this analysis highlights local
motions at Gln10–Val13, Gln22–Ser27 and Met37–
Leu40 and in the second part of helix III As could be
expected, the analysis does not reproduce exactly the
experimental findings, but it is consistent with them
overall In particular, the long loop involving Gln22–
Ser27 appears to be rather unconstrained, resulting in
large conformational motions in its central part
Simi-larly, the second part of helix III appears to be less
restrained than the first part, starting from Tyr54
Arg53 appears to be more mobile than the preceding
residues, but less free than the second part of the helix
The pattern of hydrogen bonds is consistent with
a regular a-helix throughout the simulation only for
the first part of helix III Starting from Tyr54, the
hydrogen bond with residue i-4 is not well conserved,
and for Arg53 and Tyr54, hydrogen bonds with
resi-due i-3 are also observed, in good agreement with the helix tightening suggested by NMR
Thus, the picture emerging from MD simulation is not as detailed as that provided by relaxation analysis, but it is consistent overall with the local motions observed by MF and⁄ or RSDM analysis and with pre-vious NMR structural findings
Discussion
The detailed description of the results obtained by the
MF and RSDM approaches has highlighted a crucial limitation of the MF treatment When the motions of
a protein in isotropic solution do not match the regime
of slow overall tumbling (nanoseconds) and fast local fluctuations (at most, hundreds of picoseconds), the MF-based fitting of the NMR relaxation data fails to retrieve a correct description of the dynamics As pre-viously pointed out [27], there may be three major pat-terns of deviation from the basic MF assumption that can be hardly recognized when NMR relaxation is measured with a single magnetic field MF-based fitting does not apply properly when: (a) the overall rotation is anisotropic; (b) collective motions with cor-relation time longer than 1.5–2.0 ns are present; and (c) uniform conformational exchange occurs that may
be masked by an overestimated sm For the experimen-tal data of the TTF-1 HD presented here, it was con-cluded that only the two latter causes of deviation may contribute to the erroneous estimates obtained from
MF analysis, although the possible uniform conforma-tional exchange does not involve the whole molecule, but rather specific regions We could infer this conclu-sion from the simultaneous analysis of the data obtained using the RSDM approach The fitting obtained from the correlation plots among the differ-ent spectral densities ensures that the assumption of isotropic overall tumbling is correct within the experi-mental error This is consistent with previous evidence obtained for the vnd⁄ NK-2 HD [28], which is very clo-sely related to the TTF-1 HD, as well as with explicit anisotropy calculations that rule out anisotropic motion (supplementary Doc S1) The increase in the refined overall correlation time with respect to the average value obtained from relaxation rate ratios of single residues, within the MF context, most likely arose from inclusion in the dataset of the relaxation rates with slow exchange contributions (namely those from Lys21, Gln22, Ile38, and His52) The ensuing overestimated sm, in turn, obscured the detection of exchange contributions other than those of Tyr25 (which, in fact, was excluded from the dataset for refined sm calculation) Also, the sm value of
Trang 109.85 ± 1.79 ns obtained from RSDM appears to be
too large for a 68-residue polypeptide, and suggests
the possibility of dimerization or higher-level
associa-tion An estimate of the expected sm for a compact,
globular protein, of the same molecular mass as the
domain addressed here, gives values within 6.6 and
7.6 ns [29] Although aggregation into a stable dimer
cannot be ruled out, in spite of the absence of
struc-tural evidence [4,11], the occurrence of association
equilibria also cannot be excluded a priori, although
the strong net charge of the molecule (+15) should
prevent significant association Addressing this issue
adequately, however, is beyond the scope of the
cur-rent work, and should be done in detail elsewhere
Besides the difficulty of demonstrating that the
forma-tion of a stable dimer or a labile, transient oligomer is
capable of substantially affecting the internal dynamics
of the monomers, so as to reject totally the conclusions
of this study, it is worth considering the actual
molecu-lar dimensions to account for the molecu-large sm value In
addition to the details that are discussed in
supplemen-tary Doc S1, one could mention that, as some 20
resi-dues of the TTF-1 HD appear to be statistically
disordered, the increment of the average hydrodynamic
radius is well beyond 0.05 nm, which is expected to
increase by 10% the overall sm [30] In fact, the
Stokes–Einstein relationship gives a hydrodynamic
radius of 1.98 nm for the TTF-1 HD under the
condi-tions of this study, i.e very close to the mean radius of
the NMR structure of the molecule (1.94 nm) [4] The
conclusions inferred here may be much more
intrigu-ingly challenged if one wonders whether the dynamic
properties of an isolated HD at 286 K can be extended
to the whole TTF-1 molecule under physiological
con-ditions The temperature increase at 310 K and the
molecular size of the entire transcription factor should
lead to an overall tumbling rate of 20–22 ns)1 Besides
noting that the selected experimental conditions for
characterizing the dynamics of the TTF-1 HD are not
completely unrelated to the dynamic regime within the
whole protein, it is clear that the local mobility trends
that may influence HD function should still apply, and
may possibly be elicited, under physiological
condi-tions
The most serious problem in MF interpretation of
the TTF-1 HD data can be considered to be the
cou-pling of restricted amplitudes and slow rates and,
con-versely, large amplitudes and faster rates, for the
internal motions along most of the structured core of
the molecule This picture is physically inconsistent,
and follows from the failure to account for collective
motions with correlation times > 1.5–2.0 ns [27] The
possibility that the inconsistency is due to reliability
problems with measurements at a single field rather than inherent limits of the MF framework is in con-trast to the results of interpretation of the same data obtained using the RSDM approach
Despite the limitations, even with MF analysis, peculiar local fluctuation states were recognized at Lys24, Glu30, Gln44 and Tyr54, in agreement with the corresponding spectral density mapping interpretation
In particular, it is instructive to consider the MF results obtained for Glu30 The arrangement of the helix II N-capping [4] seems to be paralleled by an increase in S2and a decrease in sefor Glu30 and, con-versely, a decrease in S2 and an increase in sefor the Arg31 15N–1H vector Thus, the result for individual
se> smi obtained for Ser27, which is involved in N-capping with Arg31 N–H and Glu30 N–H is, at least qualitatively, justified, and suggests an interpretation based on the compensation between the amplitude and frequency of local fluctuations In other words, a wider motion amplitude is accompanied by a slower motion rate because of the increased mechanical inertia
In the context of RSDM, the detailed analysis of the three spectral densities J(0), J(xN) and J(0.87xH) allowed us to obtain a rather complete description of the dynamics of the TTF-1 HD over a large range of timescales The current observations are in agreement with our previously published structural characteriza-tion of the TTF-1 HD [4] As we concluded before, the C-terminal segment of helix III, which is involved in the DNA recognition process, displays higher mobility than the preceding moiety, and Arg53 within the rec-ognition helix appears to be a hinge point Addition-ally, slow conformational exchange contributions were observed for the His52 backbone, in a ls-to-ms time-scale The high J(0) and J(xN) values obtained for the N-terminal moiety of helix III further stress its stabil-ity Within this first stretch of the recognition helix, Gln50 has a pivotal function High values of J(0) and J(xN) with a corresponding very low J(0.87xH) for the amide vector dynamics of this residue indicate local motions occurring essentially over the nanosecond timescale The lack of fast internal motions reflects the crucial role of Gln50, which behaves as mechanical point of support, needed for the hinging of the C-ter-minal part of helix III This relative rigidity of resi-due 50 is also relevant to biological function, and has been long recognized as one of the DNA recognition determinants of HD motifs [1,2]
Slow motion contributions are seen to occur for Ile38 in the hydrophobic core or for residues close to this core (i.e Lys21 and Gln22, neighboring Phe20; Tyr25, neighboring Leu26), as well as for His52 (Fig 1), because of slow conformational exchange of