Refined solution structure and backbone dynamics of the
archaeal MC1 protein
Franc¸oise Paquet, Karine Loth, Herve
´
Meudal, Franc¸oise Culard, Daniel Genest and
Ge
´
rard Lancelot
Centre de Biophysique Mole
´
culaire, CNRS UPR 4301, Orle
´
ans, France
Introduction
DNA-binding proteins play a central role in all aspects
of genetic activity within an organism, such as tran-
scription, packaging, rearrangement, replication, and
repair. Archaeons have a variety of abundant,
sequence-independent nucleoid proteins, some of which
are able to compact DNA. Among the numerous chro-
matin proteins identified in archaeons, only two –
histones and Alba homologs – are present in almost all
archaeal phyla [1].
Archaeal histones (e.g. HMfa and HMfb) are char-
acterized by an a-helical histone fold. Their monomers
are not stable, and must form homodimers. In the
presence of DNA, dimers assemble into tetramers and,
sometimes, hexamers [2]. These archaeal histone tetra-
mers wrap $ 90 bp in less than one circle, resulting in
a horseshoe-shape assembly. Histones are replaced by
other chromatin proteins in archaeons that lack them,

of the shape and nature of the binding surface, whereas the 3D structure
determined with homonuclear NMR does not. The structure features five
loops, which show a large distribution in the ensemble of 3D structures.
Evidence for the fact that this distribution signifies internal mobility on the
nanosecond time scale was provided by using
15
N-relaxation and molecular
dynamics simulations. Structural variations of the arm (11 residues)
induced large shape anisotropy variations on the nanosecond time scale
that ruled out the use of the model-free formalism to analyze the relaxation
data. The backbone dynamics analysis of MC1 was achieved by compari-
son with 20 ns molecular dynamics trajectories. Two b-bulges showed that
hydrogen bond formation correlated with u and w dihedral angle transi-
tions. These jumps were observed on the nanosecond time scale, in agree-
ment with a large decrease in
15
N-NOE for Gly17 and Ile89. One water
molecule bridging NH(Glu87) and CO(Val57) through hydrogen bonding
contributed to these dynamics. Nanosecond slow motions observed in
loops LP3 (35–42) and LP5 (67–77) reflected the lack of stable hydrogen
bonds, whereas the other loops, LP1 (10–14), LP2 (22–24), and
LP4 (50–53), were stabilized by several hydrogen bonds. Dynamics are
often directly related to function. Our data strongly suggest that residues
belonging to the flexible regions of MC1 could be involved in the interac-
tion with DNA.
Abbreviations
CSP, chemical shift perturbation; MC1, methanogen chromosomal protein 1; MD, molecular dynamics; RDC, residual dipolar coupling.
FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS 5133
HTa, a member of the HU family [5,6]. Sac7d and
Cren7 have a small b-barrel with or without an amphi-

three-dimensional structure of this architectural protein
extracted from the M. thermophila strain CHTI55 by
using
1
H-NMR spectroscopy only [15]. The overall
fold of MC1, characterized by its b–b–a–b–b–b link-
ing, is different from those of other known DNA-bind-
ing proteins. Site-directed mutagenesis showed that
two residues belonging to the loop b4–b5 (Trp74 and
Met75) are involved in DNA binding [16]. Further-
more, hydroxyl radical footprinting, together with a
dystamycin competition experiment, suggested that the
monomeric MC1 binds to DNA through the minor
groove, and that the binding site, covering at least
15 bp, is composed of two areas of contact separated
by nearly 10 bp [13]. The static structure, previously
described, could not explain this particular behavior
[15]. We therefore decided to continue the structural
study of MC1 with a recombinant
13
C,
15
N-labeled pro-
tein expressed in Escherichia coli. In this article, we
report heteronuclear NMR experiments that have
enabled us to assign all side chains and to introduce
dihedral angle restraints (u and w angles). Residual
dipolar couplings (RDCs) were also measured in a par-
tially aligned sample with radially compressed poly-
acrylamide gel to add constraints, particularly in the

another antiparallel b-bulge (B2), composed of Val57,
Glu87, and Arg88, is observed for all the structures.
The secondary structure elements are connected
by loops LP1 (10–14), LP2 (22–24), LP3 (35–42),
LP4 (50–53), and LP5 (67–77), referred to as ‘arm’ in
the text. The latter now appears to be remote from the
protein core, whereas it was previously described as
pulled down on the a-helix. Superimposition of the 15
best structures of MC1 clearly shows that the regions
with the largest degree of structural variations include
the N-terminus, C-terminus, and loops LP1, LP3, LP4,
and, especially, LP5 (Fig. 1A). Its rmsd value is large
(11 A
˚
), in agreement with the extensive conformational
space swept by its residues (Table 1), whereas the rmsd
values of the other loops fall between 2 and 2.7 A
˚
.
MC1 can no longer be considered as a spherical pro-
tein, but rather as an anisotropic structure defined by
the ratio of the principal components of the inertia
tensor. This ratio differs within the 15 models of MC1,
1.00 : (0.85–0.94) : (0.34–0.43), according to the posi-
tion of the arm.
Although this new fold is completely different from
those of other known proteins, it has similarities to the
small architectural proteins Sac7d and Cren7 belonging
to the Sulfolobus strains of the Crenarchaeota subdo-
main (Fig. 2) [3,4]. All possess a triple-stranded b-sheet

(Pro68, Pro72, Trp74, Met75, and Pro76), which are
conserved in different species of Methanosarcina and
Halobacteria. Site-directed mutagenesis showed that
two residues belonging to the loop (Trp74 and Met75)
are involved in DNA binding [16]. It is clear that the
arm of MC1 is essential for DNA binding and bend-
ing. The interaction mode of MC1 is probably com-
pletely different from those of Sac7d and Cren7, which
bind and bend DNA by placing their triple-stranded
b-sheet (b3–b4–b5) across the DNA minor groove.
Indeed, the electrostatic potential surface of MC1
reveals that one side of the protein has a considerable
number of positively charged residues: Arg4, Lys22,
Arg25, Lys53, Lys54, His56, Lys69, Arg71, Lys81,
Lys85, Lys86, and Lys91 (Fig. 1C). This side, the
reverse of the one used by Sac7d and Cren7 of Sulfolo-
bus, is a good candidate to interact with the phosphate
group of nucleotides.
15
N-NMR Relaxation for MC1
The
15
N-HSQC spectrum of MC1 recorded at
600 MHz showed good dispersion of the crosspeaks
(Fig. S1). Relaxation data were obtained for 84 back-
bone N–H pairs (93 residues minus Pro24, Pro42,
Pro68, Pro72, Pro76, Pro82, Gly51, and the two N-ter-
minal residues Ser1 and Asn2) at 600 MHz (R
1
, R

800 MHz). Such variations in R
2
values can result
from relatively large-amplitude motions, efficient
exchange processes, or shape anisotropy effects. In our
experimental conditions, no significant increase in R
2
values was observed for MC1 between 600 and
800 MHz, indicating the absence of efficient exchange
processes. We observed that R
2
values decreased sub-
stantially for Gly17, Asp66, Lys69, Asn70, Arg71, and
Ile89, whereas R
1
values increased, reflecting local
Table 1. NMR constraints and structural statistics.
NMR constraints
Distance restraints
Total NOE 1873
Unambiguous 1089
Ambiguous 784
Hydrogen bonds 37
Total dihedral angles
F 69
W 69
RDC constraints 57
Structural statistics for the ensemble of the 15 lowest-energy
structures
Average violations per structure

Disallowed (%) 0.3
F. Paquet et al. NMR structure and backbone dynamics of MC1
FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS 5135
motions. Large variations of
15
N-NOE were observed
along the sequence at 600 MHz, particularly for Thr3,
Arg4, Gly17, Asp66–Ile79, Ile89, and Glu93, for which
15
N-NOE < 0.65; these residues clearly possess con-
siderable internal motions on the nanosecond time
scale. It is interesting to locate these residues in the
structure: they belong to bulges B1 (Gly17) and B2
(Ile89), loop LP5 (Asp66, Ala67, Lys69, Asn70, Arg71,
Ala73, Trp74, Met75, Glu77, Lys78, and Ile79), and
the termini (Thr3, Arg4, and Glu93).
Although the structure of the MC1ÆDNA complex
has not yet been solved, relaxation measurements on
the complex have been conducted (Fig. S3). Besides six
Pro residues, resonance overlap precluded the interpre-
tation of relaxation data for seven residues (Phe19,
Arg25, Gly51, Asp66, Lys86, Ile89, and Glu90).
LP1
LP3
C
LP4
LP5
N
LP2
β3

code: 1AZP) and (B) Cren7 (Protein Data
Bank ID code: 3LWI) specific to Sulfolobus
(Crenarchaea). (C) MC1 (Protein Data Bank
ID code: 2KHL) specific to Methanosarcina
(Euryarchaea) and (D) HU monomer (Protein
Data Bank ID code: 1P71) specific to
Thermoplasma (Euryarchaea).
NMR structure and backbone dynamics of MC1 F. Paquet et al.
5136 FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS
Several residues belonging to arm LP5 (Ala67, Lys69,
Asn70, Ala73, Met75, and Glu77) exhibit an increase
in
15
N-NOE. If we compare the sites that exhibit back-
bone chemical shift perturbations (CSPs) upon DNA
binding with those that exhibit an increase in NOE
upon DNA binding, we can conclude that the arm
becomes much less mobile after binding with DNA
(Fig. 4). This is reminiscent of the structure and
dynamics of the highly mobile b-arms in the free pro-
tein HU, which become much less mobile after binding
with DNA. In the model proposed by Tanaka, the
DNA-binding arms can move as rigid arms, creating
sufficient room for accepting DNA [20]. The tips of
the arms are highly flexible, and once the DNA has
moved inwards, the arms close and the tips of the
arms wrap around the DNA.
The amplitudes and time scales of the intramolecular
motions experienced by the protein backbone are com-
monly determined from the

changes over time, its associated rotational diffusion
tensor varies. The eigenvalues of the diffusion tensor
2.5
A
B
C
β1 β2 β3α1 β4 β5
0.5
1.0
1.5
2.0
R
1
(s
–1
)R
2
(s
–1
)15
N-NOE
0.0
10
15
20
0
5
1
0
0.2

), as
indicated by the distance C
a
–C
a
between Val65 at the
end of strand b4 and Arg71 at the extremity of
arm LP5. This stretch was made up of complex motions
in the arm, as shown by the variations in C
a
–C
a
dis-
tances between Ala67–Arg71, Ala67–Lys78, Arg71–
Val65, and Arg71–Ile79. The motion of the arm is cen-
tered on a hinge composed of Ala67 and Glu77. More-
over, loops LP1 and LP3 exhibited substantial
conformational changes during the trajectory, as
shown by variations in the C
a
–C
a
distances between
Glu11–Asp43, Gly13–Leu92, and Gly35–Lys62. Dur-
ing the trajectory, the location of loop LP1 changed in
relation to strands b3 and b5, as indicated by varia-
tions in the Glu11–Asp43 and Gly13–Leu92 distances.
Internal correlation functions
The internal autocorrelation functions are calculated
within the molecular reference frame of the superposed

in blue, and those with an intermediate
increase are in marine. (B) Residues that
exhibit significant CSP upon DNA binding
are in red, and those with intermediate
changes are in orange.
1.9
1.3
1.5
1.7
D
x
(10
7
s
–1
)
D
y
(10
7
s
–1
)
D
z
(10
7
s
–1
)

correlation time of 8.6 ± 0.3 ns calculated using
hydronmr during the trajectory.
The MD-derived order parameters S
2
values for the
b-strands and the a-helix are consistent with the exper-
imental relaxation data (Fig. 7B). Slow motions were
detected for Ser1, Asn2, Leu92, Glu93 (terminal resi-
dues), Gly17 to Phe19 (bulge B1), Arg34 to Gly37
(loop LP3), Gly51 and Thr52 (loop LP4), Ala67 to
Lys78 (loop LP5), and Glu87 to Ile89 (bulge B2). The
largest amplitudes were observed for Ser36 (S
2
= 0.13)
and from Ala67 to Asn70 (0.1 < S
2
< 0.16). The resi-
dues involved in the two bulges have S
2
values around
0.6, which is consistent with the
15
N-NOE values. The
calculated S
2
values in the loops are lower than
expected, particularly in loops LP3 and LP4. A recent
study provides evidence for a specific link between
force field deficiencies and disagreement between
experimental and MD order parameters [26]. MD sim-

16
71–65
6
8
10
12
8
10
35–62
10
12
14
9
11
71–67
Distance (
Å
)
Distance (Å)
4
6
8
12
14
11–43
5
7
12
14
71–79

Distance (Å)
92
13
11
43
35
62
79
78
65
67
71
92
13
11
43
35
62
79
78
65
67
71
A
B
Fig. 6. (A) Some C
a
–C
a
distances (A

explain the greater flexibility of this bulge and the slow
internal motions of Glu87, Arg88 and Ile89 with very
large amplitude. These motions were correlated with
the dihedral angle transitions of w(Glu87), u(Arg88),
and u(Ile89). The presence of these hydrogen bonds
was consistent with the homonuclear NOEs found in
this region [15].
Loop LP1 was stabilized with two hydrogen bonds,
NH(Asp10)–CO(Asn14) and NH(Gly13)–CO(Asp10),
throughout the trajectory time. Similarly, the hydrogen
bond NH(Gln26)–CO(Gln23) stabilized the short loop
LP2 for 19 ns.
The lack of stable hydrogen bonds in loop LP3 (35–
42) corresponds with large motions of the N)H
vectors for Gly35, Ser36, and Gly37. However, this
nonstructured loop is probably not important in the
DNA binding, because the number of residues between
Gly35 and Ile45 (MC1-CHTI55 numbering) varies
from 3 to 14 in different species of Halobacteria and
Methanomicrobia [15].
Loop LP4 (50–53) was stabilized by two hydrogen
bonds, NH(Thr52)–CO(Glu49) and NH(Leu92)–
CO(Lys53), binding the loop to strand b5 for 11 ns.
Supplementary hydrogen bonds involving the side
chains NH
2
(Arg48)–CO(Thr52) and OH(Thr52)–
CO(Glu49) contribute to the stiffness of the structure
for a short time. Moreover, two NOE crosspeaks,
OH(Thr52)–NH(Thr52) and OH(Thr52)–NH(Glu49),

0.2
0.4
0123
45
678910
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91
S
2
Sequence
Fig. 7. (A) Three representative internal cor-
relation functions computed on a trajectory
of 20 ns for Gln26 in the a-helix, Val18 in a
bulge, and Asn70 in the arm. (B) Residue
profile of the MD-derived S
2
.
NMR structure and backbone dynamics of MC1 F. Paquet et al.
5140 FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS
corresponds to a combination of slow motions of
large amplitude. In accordance with this, only four
hydrogen bonds were observed: NH(Lys71)–CO
(Pro68), NH
2
(Lys71)–CO(Pro76), NH(Met75)–CO
(Pro72), and NH(Trp74)–CO(Pro72) for 6.5, 11, 16
and 0.3 ns respectively.
Summary
In summary, the structure of MC1, consisting of a
pseudobarrel with an extension of the b-sheet (b4–b5)
forming an arm of 11 residues, has been refined. The

A
100
–50
0
100
200
300
Phi angle (deg)
Phi angle (deg)Phi angle (deg)
G17
50
150
250
R88
100
200
300
Psi angle (deg)
V18
50
100
150
200
I89
7.0
NH8 - CO16
8.0
NH57 - CO87
1.0
3.0

of the bulges. The dotted lines and the time
characterized the presence of hydrogen
bonding during the trajectory.
F. Paquet et al. NMR structure and backbone dynamics of MC1
FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS 5141
Experimental procedures
Preparation of
13
C,
15
N-labeled MC1
The proteins were expressed in BL-21(DE3) cells trans-
formed with the pET24a–mc1 plasmid. Protein doubly
labeled with
15
N and
13
C was obtained by using an iso-
tope-enriched Celtone-rich medium (Martek Biosciences,
Columbia, MD, USA). To obtain
15
N-labeled protein,
cells were first grown in LB medium, and then, at a
D
600 nm
of 0.7, they were collected and resuspended in
M9 medium containing
15
NH
4

from Eurogentec (Lie
`
ge, Belgium) (OliGold oligonucleotides
quality). The single-stranded 15 bp oligodeoxynucleotides
were characterized by NMR and annealed at a 1 : 1 ratio.
The MC1ÆDNA complex was prepared by slowly adding
the 7.5 mm DNA duplex solution (10 mm phosphate buffer,
pH 6, 100 mm NaCl, 1 mm EDTA, 10% D
2
O) to the
1.6 mm protein solution (10 mm phosphate buffer, pH 6,
100 mm NaCl, 1 mm EDTA, 10% D
2
O) to give a final
complex concentration of $ 1mm.
NMR spectroscopy and structure calculations
Two-dimensional and three-dimensional NMR experiments
were performed on a 600 MHz Varian
UNITY
INOVA spec-
trometer at 299 K. Spectra were processed with nmrpipe
[28], and analyzed with nmrview [29]. Backbone and side
chain resonance assignments were obtained from the stan-
dard triple resonance experiments [30]. 4,4-dimethyl-4-sila-
pentane-1-sulfonic acid was used as a
13
C chemical shift
reference. Interproton distances were derived from NOESY
datasets obtained at mixing times of 100, 150 and 200 ms.
Backbone dihedral angle restraints were determined with

was calculated by using the pdb2pqr server (version 1.6)
[36] and apbs software [37]. The figures were prepared with
pymol [38] or molmol [39].
Determination and analysis of
15
N-relaxation
parameters (R
1
, R
2
, and NOE) for MC1
NMR relaxation experiments were measured at 299 K on
a Varian 500 MHz (NOE), Varian INOVA 600 MHz
(NOE, R
1
and R
2
) and Varian INOVA 800 MHz (R
1
and
R
2
) equipped with a cryogenic triple resonance probe spec-
trometer. On each instrument,
15
N R
1
and R
2
spectra were

⁄ I
eq
, where I
sat
and I
eq
were
the volumes of a crosspeak in the spectra collected with
and without proton saturation. Both were acquired with
64 scans. All experiments were run twice in the same con-
ditions. Volumes for the amide
15
N–
1
H crosspeaks were
measured by using nmrview software [29]. Uncertainties
in the volumes were measured from the duplicate spectra.
After obtaining volumes of crosspeaks and their errors,
the above time series were fitted from a single exponential
decay function.
Relaxation experiments for the MC1ÆDNA complex were
performed at 600 MHz as described above, with R
1
relaxa-
tion delays of 10, 100, 200, 300, 500, 800, 1000 and
1300 ms and R
2
relaxation delays of 10, 30, 50, 70, 90, 110,
130 and 150 ms at 299 K.
Backbone CSPs and

mization, and MD simulation [41].
A total of 2000 snapshots with a time increment of 10 ps
were analyzed from the final 20 ns of the MD simulation.
Prior to this, overall translational and reorientational
motions were removed by a least squares superposition of
the secondary structure backbone atoms of each snapshot
on those of the mean snapshot. The mean structure is taken
as the most central structure among the simulated ones,
and corresponds to the one at 12.01 ns in the present work.
During the analysis, the rmsd between two structures was
evaluated as:
rmsd ¼
1
N

X
ðr
1
À r
2
Þ
2

1
2
where N is the number of atoms taken into consideration,
r
1
and r
2

where P
2
[x] is the second Legendre polynomial, and l is the
N–H bond vector scaled to unit magnitude.
When the internal correlation function is made up of
three decreasing exponentials, the expression of the internal
correlation function is:
CðtÞ¼S
2
þ A
f
e
ðÀt=s
f
Þ
þ A
m
e
ðÀt=s
m
Þ
þ A
s
e
ðÀt=s
s
Þ
with A
f
, A

Þ
þð1 À S
2
s
ÞS
2
f
S
2
m
e
ðÀt=s
s
Þ
where S
2
f
¼ 1 À A
f
; S
2
m
¼ð1 À A
f
À A
m
Þ=ð1 À A
f
Þ; S
2

), and finally
to a third plateau (S
2
).
Hydrogen bonding
A hydrogen bond was considered to be present when the
distance between the donor (D) and the acceptor (A) was
smaller than 3.5 A
˚
and the D–H–A angle was larger than
135°.
Acknowledgements
The authors would like to thank S. Goffinont (CBM,
Orle
´
ans, France) for his participation in preparing the
labeled MC1. Financial support from the TGE RMN
THC Fr3050 for conducting the research is gratefully
acknowledged.
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Supporting information
The following supplementary material is available:
Fig. S1. Six hundred megahertz
1
H–
15
N-HSQC NMR
spectrum of MC1 (1.6 mm), 800 mm NaCl, and
100 mm acetate buffer (pH 5.1, 26 °C). Peaks corre-
sponding to the NH
2
groups of the side chain amides
of Asp and Gln residues are connected by thin lines.
Fig. S2. Backbone
15
N-relaxation data for 1.6 mm free
MC1 at 800 MHz: (A) Longitudinal relaxation rate.
(B) Transverse relaxation rate.

should be addressed to the authors.
F. Paquet et al. NMR structure and backbone dynamics of MC1
FEBS Journal 277 (2010) 5133–5145 ª 2010 The Authors Journal compilation ª 2010 FEBS 5145