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Electronic supplementary material
Solution structure of the C-terminal domain of multiprotein bridging
factor 1 (MBF1) of Trichoderma reesei
Roberto K. Salinas1*, Cesar M. Camilo1, Simona Tomaselli2, Estela Y. Valencia1, Chuck S.
Farah1, Hamza El-Dorry1,3 and Felipe S. Chambergo4*
1. Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes 748, 05508-900,
São Paulo SP, Brazil.
2. Laboratorio NMR, ISMAC, CNR, via Bassini 15, 20133, Milano, Italy.
3. Department of Biology and the Science and Research Technology Center, The American
University in Cairo, 113 Kasr El Aini Street, Cairo, Egypt.
4. School of Arts, Sciences and Humanities, University of São Paulo, Av. Arlindo Bettio
1000, 03828-000, São Paulo SP, Brazil.
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Nterminal
Cterminal
I
II
III
IV
2
A
RMSD
1.5
1
0.5
0
50
I
60
70
80
90
100
110
120
130
140
150
160
Residue number
Nterminal
Number of constraints
B
Cterminal
I
II
III
IV
60
40
20
0
68
88
108
128
148
Residue number
Figure S1. Ctd-TrMBF1 structure statistics. (A) Per-residue root mean square deviation
(rmsd) for the C, CO and N atoms coordinates from residues 78-148 with respect to the
mean. (B) Distribution of intra-residual, sequential, short range and long range NOEs
(white, shaded, dark shaded and black, respectively) along the protein sequence. The
positions of the secondary structure elements in Ctd-TrMBF1 are indicated at the top.
2
I
II
III
IV
6
5
4
logP
3
2
1
0
50
60
70
80
90
100 110 120 130 140 150
Residue number
Figure S2. Hydrogen-deuterium exchange protection factors as a function of the
amino acid sequence. The positions of the alpha-helices are indicated at the top. All amide
protons located at the termini exchanged too fast to be monitored in this experiment. The
exceptions are the amide protons of Phe94 and Met98 in loop I, Glu109 in the beta-turn, Val138
and Leu140 in the beginning of the C-terminal loop. The reasons for these high protection
factors can be found analyzing the NMR structures: Glu109 HN, in the beta-turn, donates a
hydrogen bond to the backbone carbonyl group of Gly104 in helix II in all 20 structures of
the NMR ensemble. In the first loop, Phe94 HN and Met98 HN donate a hydrogen bond to
Arg91 CO in six and 17 structures, respectively. In the C-terminal loop, Val138 HN donate a
hydrogen bond to Glu133 backbone carbonyl oxygen, while Leu140 HN donate a hydrogen
bond to its side chain O in 11 structures. This experiment was performed at 283 K, pH
3
meter reading 7.07 and at the 500 MHz spectrometer. The data analysis for the calculation
of protection factors was done as described previously (Salinas et al. 2006. Protein Sci. 15
1752-1759).
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Figure S3. Structural comparison with EDF1. (A) The most representative models of the
NMR ensemble of EDF1 (PDB 1x57, unpublished) (blue) and Tr-CtdMBF1 (yellow) were
superimposed over the coordinates of the backbone atoms of the four alpha-helices. (B)
Sequence comparison of MBF1 from different organisms. The conserved Arg91 is shown in
grey. The positions of the alpha-helices and of the C-terminal loop of Ctd-TrMBF1 are
shown at the top. (C) On the right, the different orientations of Arg91 side chain (ball and
sticks) in EDF1 (blue) and Ctd-TrMBF1 (yellow). The nitrogen atoms (N, N1, N2) are
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represented by blue spheres. The C carbon of Ile87 in helix I of Ctd-TrMBF1 is colored
red. The side chain of Glu118 in EDF1 is shown in ball and sticks, with the O atoms as red
spheres. Numbering of -helices are indicated by arrows. In Ctd-TrMBF1 the Arg91N
donates a hydrogen bond to Ile87 CO in 11 structures and the guanidinium group is buried
within the hydrophobic core. In EDF1, however, the orientation is different and the N
donates a hydrogen bond to the Ooxygens of Glu118 in helix III. On the left a region of the
2D NOESY spectrum is shown. Three long range NOEs define the proximity between
Arg91H and Ile87 side chains in Ctd-TrMBF1. The illustrations were prepared with
Molmol (Koradi R, Billeter M, Wüthrich K. MOLMOL: a program for display and analysis
of macromolecular structures. J Mol Graphics 1996; 14: 51-55).
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A)
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B)
Figure S4. The electrostatic potential at the solvent accessible surface of 434 repressor,
Ctd-TrMBF1 and hEDF1. (A) Solvent accessible surface representations of Ctd-TrMBF1,
phage 434 repressor (PDB ID 1PRA) and hEDF1 (PDB ID 1X57), colored according to the
electrostatic surface potential in the range of +2 kT/e (blue) to -2 kT/e (red). The
calculations of the electrostatic surface potential were performed using a ABPS (Baker et al
Proc Natl Acad Sci USA 2001; 98: 10037-10041) plug-in for Pymol written by M. Lerner
(http://www.pymolwiki.org/index.php/APBS) , and assuming salt concentration of 0.06 M
and dielectric constants of 2 and 80 for the protein interior and the water, respectively, and
temperature of 298 K. The structures are oriented such that helix III is placed horizontally
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and in the direction of the reader. This helix is the only one which is not indicated by its
number. On the right, the surface representations were made partially transparent in order
to show the ribbon representations at the backbone. The comparison shows that the
electrostatic surface potentials at helix III tend to be less positive in Ctd-TrMBF1 and
hEDF1 than in the 434 repressor. This observation is consistent with the fact that the third
helix contains more hydrophobic residues in Ctd-TrMBF1 and hEDF1 than in 434
repressor, especially at their N-termini (helix III residues are AATVASY, PQVIADY and
QQSIEQL for Ctd-TrMBF1, hEDF1 and 434 repressor, respectively). (B) Comparison of
the DNA binding surface of phage 434 repressor with the corresponding surface of CtdTrMBF1, assuming that it would recognize and bind DNA in the same manner as the 434
repressor. At the top, the phage 434 repressor is shown bound to the left side of the 434
DNA operator (PDB ID 1PER); the second repressor molecule was omitted in order to
make the figure more clear. At the bottom, the Ctd-TrMBF1 NMR structure was rotated to
the same orientation of phage 434 repressor, and was superimposed over the coordinates of
the 434 DNA operator from the same PDB entry. On the left the surface representations are
solid, while on the right they were made transparent to show the ribbom representations.
Helices I, II and IV are marked. The DNA is shown as a sticks representation. The proteins’
solvent accessible surfaces are colored according to their electrostatic surface potentials as
in item A. In the 434 repressor the surface of the loop connecting helices III and IV, which
contributes with important contacts to the DNA phosphates, including Arg43 which inserts
into the minor groove (see for example Anderson et al. 1987 Nature 326 846-852), is
characterized by positive electrostatic potentials. The equivalent region in Ctd-TrMBF1 has
negative electrostatic potential, and perhaps this would be unfavorable for DNA binding
and could explain why other studies did not detect MBF1 DNA binding activity.
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