Pages 1–14
2v5w
Evolutionary trace report by report maker
May 12, 2010
4.4
4.5
4.6
4.7
1
CONTENTS
1
Introduction
1
2
Chain 2v5wB
2.1 Q9BY41 overview
2.2 Multiple sequence alignment for 2v5wB
2.3 Residue ranking in 2v5wB
2.4 Top ranking residues in 2v5wB and their position on
the structure
2.4.1 Clustering of residues at 25% coverage.
2.4.2 Overlap with known functional surfaces at
25% coverage.
2.4.3 Possible novel functional surfaces at 25%
coverage.
1
1
1
2
10
3
Notes on using trace results
3.1 Coverage
3.2 Known substitutions
3.3 Surface
3.4 Number of contacts
3.5 Annotation
3.6 Mutation suggestions
12
12
12
12
13
13
13
4
Appendix
4.1 File formats
4.2 Color schemes used
4.3 Credits
13
13
13
13
2
2
3
4.3.1 Alistat
4.3.2 CE
4.3.3 DSSP
4.3.4 HSSP
4.3.5 LaTex
4.3.6 Muscle
4.3.7 Pymol
Note about ET Viewer
Citing this work
About report maker
Attachments
13
13
14
14
14
14
14
14
14
14
14
INTRODUCTION
From the original Protein Data Bank entry (PDB id 2v5w):
Title: Crystal structure of hdac8-substrate complex
Compound: Mol id: 1; molecule: histone deacetylase 8; synonym:
hdac8, hd8; chain: a, b; mutation: yes; engineered: yes; mol id: 2;
molecule: peptidic substrate; chain: i, l; engineered: yes; mol id:3;
molecule: glycyl-glycyl-glycine; chain: g; engineered: yes
Organism, scientific name: Homo Sapiens;
2v5w contains a single unique chain 2v5wB (367 residues long)
and its homologue 2v5wA. Chains 2v5wG, 2v5wI, and 2v5wL are
too short to permit statistically significant analysis, and were treated
as a peptide ligands.
2 CHAIN 2V5WB
2.1 Q9BY41 overview
From SwissProt, id Q9BY41, 96% identical to 2v5wB:
Description: Histone deacetylase 8 (HD8).
Organism, scientific name: Homo sapiens (Human).
Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata;
Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates;
Catarrhini; Hominidae; Homo.
Function: Responsible for the deacetylation of lysine residues on
the N-terminal part of the core histones (H2A, H2B, H3 and H4).
Histone deacetylation gives a tag for epigenetic repression and plays
an important role in transcriptional regulation, cell cycle progression
and developmental events. Histone deacetylases act via the formation
of large multiprotein complexes.
Subunit: Interacts with PEPB2-MYH11, a fusion protein consisting
of the 165 N-terminal residues of CBF-beta (PEPB2) with the tail
region of MYH11 produced by the inversion Inv(16)(p13q22), a
translocation associated with acute myeloid leukemia of M4EO subtype. The PEPB2-MYH1 fusion protein also interacts with RUNX1,
a well known transcriptional regulator, suggesting that the interaction
with HDAC8 may participate to convert RUNX1 into a constitutive
transcriptional repressor.
Subcellular location: Nuclear; excluded from the nucleoli.
1
Lichtarge lab 2006
Alternative products:
Event=Alternative splicing; Named isoforms=3; Name=1;
IsoId=Q9BY41-1; Sequence=Displayed; Name=2; IsoId=Q9BY412; Sequence=VSP 007176, VSP 007177; Note=Derived from EST
data; Name=3; IsoId=Q9BY41-3; Sequence=VSP 007174, VSP
007175;
Tissue specificity: Weakly expressed in most tissues. Expressed at
higher level in heart, brain, kidney and pancreas.
Miscellaneous: Its activity is inhibited by trichostatin A (TSA) and
butyrate, two well known histone deacetylase inhibitors.
Similarity: Belongs to the histone deacetylase family. Type 1
subfamily.
About: This Swiss-Prot entry is copyright. It is produced through a
collaboration between the Swiss Institute of Bioinformatics and the
EMBL outstation - the European Bioinformatics Institute. There are
no restrictions on its use as long as its content is in no way modified
and this statement is not removed.
2.2
Fig. 1. Residues 10-192 in 2v5wB colored by their relative importance. (See
Appendix, Fig.15, for the coloring scheme.)
Multiple sequence alignment for 2v5wB
For the chain 2v5wB, the alignment 2v5wB.msf (attached) with 341
sequences was used. The alignment was downloaded from the HSSP
database, and fragments shorter than 75% of the query as well as
duplicate sequences were removed. It can be found in the attachment
to this report, under the name of 2v5wB.msf. Its statistics, from the
alistat program are the following:
Format:
MSF
Number of sequences: 341
Total number of residues:
Smallest:
142
Largest:
367
Average length:
335.9
Alignment length:
367
Average identity:
43%
Most related pair:
99%
Most unrelated pair: 0%
Most distant seq:
42%
Fig. 2. Residues 193-376 in 2v5wB colored by their relative importance. (See
Appendix, Fig.15, for the coloring scheme.)
114539
Pymol script for producing this figure can be found in the attachment.
Furthermore, <1% of residues show as conserved in this alignment.
The alignment consists of 29% eukaryotic ( 6% vertebrata, <1%
arthropoda, 12% fungi, 4% plantae), 3% prokaryotic, and <1%
archaean sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be
found in the attachment, under the name 2v5wB.descr.
2.3
Residue ranking in 2v5wB
The 2v5wB sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues
in 2v5wB can be found in the file called 2v5wB.ranks sorted in the
attachment.
2.4
Top ranking residues in 2v5wB and their position on
the structure
In the following we consider residues ranking among top 25% of
residues in the protein . Figure 3 shows residues in 2v5wB colored
by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A
Fig. 3. Residues in 2v5wB, colored by their relative importance. Clockwise:
front, back, top and bottom views.
2
2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the
top 25% of all residues, this time colored according to clusters they
belong to. The clusters in Fig.4 are composed of the residues listed
3) suggests possible disruptive replacements for these residues (see
Section 3.6).
Fig. 4. Residues in 2v5wB, colored according to the cluster they belong to:
red, followed by blue and yellow are the largest clusters (see Appendix for
the coloring scheme). Clockwise: front, back, top and bottom views. The
corresponding Pymol script is attached.
res
type
178
D
180
H
151
G
153
C
142
H
209
P
143
H
140
G
152
F
267
D
305
G
101
D
208
F
303
G
304
G
207
F
in Table 1.
cluster
color
red
size
86
Table 1.
member
residues
37,44,70,71,75,79,98,101,102
103,105,136,137,139,140,141
142,143,144,149,150,151,152
153,154,156,157,159,162,165
171,172,173,174,176,178,179
180,181,182,183,184,185,186
188,189,192,195,196,197,199
201,202,207,208,209,210,211
212,216,218,222,228,230,231
237,257,263,265,266,267,268
269,272,273,274,275,277,280
302,303,304,305,306,313,315
Table 1. Clusters of top ranking residues in 2v5wB.
subst’s
(%)
D(97)S.
TG
H(97)SL
.TPD
G(98)R.
WS
C(95)
S(2)R.G
N
H(96)L
.(1)SRP
Y
P(96)F
.(1)KSR
Q
H(96)QA
.(1)NS
G(95)
.(1)SR
P(1)
F(96)
Y(1)H.S
LA
D(94)
.(3)HNG
SFT
G(93)
.(4)ASC
NF
D(86)
G(2)
E(3)
.(4)T
S(2)PKN
F(88)IP
Y(3)
.(1)
W(4)LGX
G(92)
.(4)LRH
E(1)A
G(92)
.(4)YER
KSDN
F(52)
Y(29)
L(9)N
Table 2.
cvg
0.00
noc/
bb
9/0
dist
(Å)
3.62
antn
site
0.01
24/0
3.45
site
0.03
18/18
3.01
0.04
1/0
4.33
0.05
6/0
4.03
0.05
17/17
3.29
0.06
11/0
3.73
0.07
1/1
4.96
0.07
36/1
3.56
site
0.07
6/0
2.94
site
0.10
1/1
4.95
0.11
29/0
3.11
0.12
70/8
3.40
0.13
5/5
4.19
0.14
7/7
3.30
0.16
6/6
4.35
site
continued in next column
2.4.2 Overlap with known functional surfaces at 25% coverage.
The name of the ligand is composed of the source PDB identifier
and the heteroatom name used in that file.
Interface with the peptide 2v5wL. Table 2 lists the top 25% of
residues at the interface with 2v5wL. The following table (Table
3
Table 2. continued
res type subst’s
(%)
I(1)
.(1)
R(2)APV
W
306
F
Y(91)
.(5)KLF
NHW
141
W
L(70)
M(6)
T(4)
R(5)
W(5)S
.(2)K
F(1)
G(1)YN
210
G
G(85)
R(1)
E(1)P
C(1)
A(1)
.(1)LY
F(2)
K(1)NQS
DV
274
M
I(3)
L(80)
.(3)
H(1)V
F(2)
Y(1)
Q(1)
M(3)ASC
97
G
N(54)
S(2)
G(27)
.(6)
L(2)
D(1)FAH
IKTQC
cvg
noc/
bb
dist
(Å)
0.17
10/0
3.70
0.18
5/0
3.56
0.18
10/10
3.94
Table 3. continued
res type disruptive
mutations
153
C
(E)(R)(K)(FW)
H
(E)(T)(D)(Q)
142
P
(Y)(T)(R)(H)
209
143
H
(E)(TD)(M)(Q)
G
(E)(KR)(FWHD)(QM)
140
F
(K)(E)(Q)(D)
152
D
(R)(FKWH)(Y)(VQMA)
267
305
G
(KER)(QH)(D)(M)
D
(R)(FWH)(Y)(K)
101
F
(K)(E)(Q)(R)
208
303
G
(E)(KR)(D)(FWH)
G
(R)(FW)(K)(E)
304
F
(E)(K)(T)(D)
207
306
F
(E)(K)(T)(D)
W
(E)(K)(D)(T)
141
G
(R)(KE)(H)(FW)
210
M
(Y)(H)(TR)(SCG)
274
97
G
(R)(E)(K)(H)
antn
Table 3. List of disruptive mutations for the top 25% of residues in
2v5wB, that are at the interface with 2v5wL.
0.20
4/0
3.76
0.25
6/6
3.83
Table 2. The top 25% of residues in 2v5wB at the interface with 2v5wL.
(Field names: res: residue number in the PDB entry; type: amino acid type;
substs: substitutions seen in the alignment; with the percentage of each type
in the bracket; noc/bb: number of contacts with the ligand, with the number of
contacts realized through backbone atoms given in the bracket; dist: distance
of closest apporach to the ligand. )
res
178
180
151
Fig. 5. Residues in 2v5wB, at the interface with 2v5wL, colored by their relative importance. 2v5wL is shown in backbone representation (See Appendix
for the coloring scheme for the protein chain 2v5wB.)
Table 3.
disruptive
mutations
D
(R)(FWH)(K)(QM)
H
(E)(Q)(K)(M)
G
(E)(K)(D)(R)
continued in next column
type
Figure 5 shows residues in 2v5wB colored by their importance, at the
interface with 2v5wL.
Interface with the peptide 2v5wG. Table 4 lists the top 25% of
residues at the interface with 2v5wG. The following table (Table
5) suggests possible disruptive replacements for these residues (see
Section 3.6).
4
res
type
183
D
209
P
143
H
208
F
150
S
Table 4.
subst’s
cvg
(%)
D(95)T
0.02
N(2)Q.G
P(96)F
0.05
.(1)KSR
Q
H(96)QA 0.06
.(1)NS
F(88)IP 0.12
Y(3)
.(1)
W(4)LGX
S(80)
0.23
T(1)
N(3)
A(5)
G(3)EYF
.HRMDCV
noc/
bb
4/0
dist
(Å)
4.66
8/6
3.97
1/1
4.63
6/0
3.42
7/0
4.07
Table 4. The top 25% of residues in 2v5wB at the interface with 2v5wG.
(Field names: res: residue number in the PDB entry; type: amino acid type;
substs: substitutions seen in the alignment; with the percentage of each type
in the bracket; noc/bb: number of contacts with the ligand, with the number of
contacts realized through backbone atoms given in the bracket; dist: distance
of closest apporach to the ligand. )
res
type
183
209
143
208
150
D
P
H
F
S
Table 5.
disruptive
mutations
(R)(FWH)(Y)(K)
(Y)(T)(R)(H)
(E)(TD)(M)(Q)
(K)(E)(Q)(R)
(KR)(QH)(FMW)(E)
Table 5. List of disruptive mutations for the top 25% of residues in
2v5wB, that are at the interface with 2v5wG.
Figure 6 shows residues in 2v5wB colored by their importance, at the
interface with 2v5wG.
Potassium ion binding site. Table 6 lists the top 25% of residues
at the interface with 2v5wBK1378 (potassium ion). The following
table (Table 7) suggests possible disruptive replacements for these
residues (see Section 3.6).
5
res
type
189
F
197
T
Table 6.
subst’s
cvg noc/
dist antn
(%)
bb
(Å)
F(96)Y
0.06 4/3 2.59 site
L(1).MA
H
T(88)G
0.11 1/1 4.67
V(1)
I(1)S
continued in next column
acid type; substs: substitutions seen in the alignment; with the percentage of
each type in the bracket; noc/bb: number of contacts with the ligand, with
the number of contacts realized through backbone atoms given in the bracket;
dist: distance of closest apporach to the ligand. )
res
type
189
197
222
195
192
196
F
T
G
V
T
M
Table 7.
disruptive
mutations
(K)(E)(T)(Q)
(R)(K)(H)(FW)
(R)(KE)(H)(FW)
(E)(KR)(Y)(D)
(R)(K)(H)(FW)
(Y)(H)(R)(T)
Table 7. List of disruptive mutations for the top 25% of residues in
2v5wB, that are at the interface with potassium ion.
Fig. 6. Residues in 2v5wB, at the interface with 2v5wG, colored by their relative importance. 2v5wG is shown in backbone representation (See Appendix
for the coloring scheme for the protein chain 2v5wB.)
Table 6. continued
res type subst’s
(%)
Y(2)
M(1)
L(1).QC
K
222
G
G(83)
S(1)
.(4)E
A(5)
N(1)VCY
RPQ
195
V
V(92)R
I(2)L
T(1)
C(1).FA
192
T
T(62)
N(2)V
S(15)
D(14)PC
R(1)E.L
F
196
M
M(62)
L(22)
F(6)
Q(1)A
C(1)TVR
P.SYIX
cvg
noc/
bb
dist
(Å)
0.17
2/2
4.59
0.19
3/3
2.64
0.21
5/4
2.88
0.24
3/3
4.42
antn
Fig. 7. Residues in 2v5wB, at the interface with potassium ion, colored by
their relative importance. The ligand (potassium ion) is colored green. Atoms
further than 30Å away from the geometric center of the ligand, as well as on
the line of sight to the ligand were removed. (See Appendix for the coloring
scheme for the protein chain 2v5wB.)
site
Figure 7 shows residues in 2v5wB colored by their importance, at the
interface with 2v5wBK1378.
MCM binding site. Table 8 lists the top 25% of residues at
the interface with 2v5wLMCM6 (mcm). The following table (Table
9) suggests possible disruptive replacements for these residues (see
Section 3.6).
Table 6. The top 25% of residues in 2v5wB at the interface with potassium ion.(Field names: res: residue number in the PDB entry; type: amino
6
res
type
152
F
101
D
274
M
subst’s
(%)
F(96)
Y(1)H.S
LA
D(86)
G(2)
E(3)
.(4)T
S(2)PKN
I(3)
L(80)
.(3)
H(1)V
F(2)
Y(1)
Q(1)
M(3)ASC
Table 8.
cvg
0.07
noc/
bb
14/0
dist
(Å)
3.53
antn
site
0.11
9/0
2.97
site
0.20
1/0
4.79
Table 8. The top 25% of residues in 2v5wB at the interface with
MCM.(Field names: res: residue number in the PDB entry; type: amino acid
type; substs: substitutions seen in the alignment; with the percentage of each
type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist:
distance of closest apporach to the ligand. )
res
type
152
101
274
F
D
M
Fig. 8. Residues in 2v5wB, at the interface with MCM, colored by their relative importance. The ligand (MCM) is colored green. Atoms further than 30Å
away from the geometric center of the ligand, as well as on the line of sight
to the ligand were removed. (See Appendix for the coloring scheme for the
protein chain 2v5wB.)
Table 9.
disruptive
mutations
(K)(E)(Q)(D)
(R)(FWH)(Y)(K)
(Y)(H)(TR)(SCG)
Table 10. continued
res type subst’s
(%)
.(1)NS
267
D
D(94)
.(3)HNG
SFT
304
G
G(92)
.(4)YER
KSDN
179
L
I(29)
V(39)Y
A(13)
L(10)
C(1)
M(2).GS
FPH
306
F
Y(91)
.(5)KLF
NHW
Table 9. List of disruptive mutations for the top 25% of residues in
2v5wB, that are at the interface with MCM.
Figure 8 shows residues in 2v5wB colored by their importance, at the
interface with 2v5wLMCM6.
Zinc ion binding site. Table 10 lists the top 25% of residues at the
interface with 2v5wBZN1379 (zinc ion). The following table (Table
11) suggests possible disruptive replacements for these residues (see
Section 3.6).
res
type
178
D
180
H
142
H
143
H
Table 10.
subst’s
cvg noc/
dist antn
(%)
bb
(Å)
D(97)S. 0.00 6/2 1.96 site
TG
H(97)SL 0.01 8/2 2.07 site
.TPD
H(96)L
0.05 2/0 4.35
.(1)SRP
Y
H(96)QA 0.06 1/0 4.73
continued in next column
cvg
noc/
bb
dist
(Å)
antn
0.07
4/0
1.95
site
0.14
2/2
4.12
0.15
4/3
4.25
0.17
1/0
4.79
Table 10. The top 25% of residues in 2v5wB at the interface with zinc
ion.(Field names: res: residue number in the PDB entry; type: amino acid
type; substs: substitutions seen in the alignment; with the percentage of each
type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist:
distance of closest apporach to the ligand. )
7
res
type
178
180
142
143
267
304
179
306
D
H
H
H
D
G
L
F
Table 12. continued
res type subst’s
(%)
89
D
61.(10)
61
275
C
G(72)
.(3)
S(4)
K(2)
T(4)
A(6)
N(1)
C(2)R
V(1)EIH
202
K
K(63)
Q(6)
G(2)
R(6)C
L(1)
H(2)
E(12)
.(1)
A(1)
M(1)PNF
S
207
F
F(52)
Y(29)
L(9)N
I(1)
.(1)
R(2)APV
W
306
F
Y(91)
.(5)KLF
NHW
273
P
R(61)
L(1)
K(6)
.(3)YT
P(22)VE
HDCMG
Table 11.
disruptive
mutations
(R)(FWH)(K)(QM)
(E)(Q)(K)(M)
(E)(T)(D)(Q)
(E)(TD)(M)(Q)
(R)(FKWH)(Y)(VQMA)
(R)(FW)(K)(E)
(R)(Y)(K)(H)
(E)(K)(T)(D)
Table 11. List of disruptive mutations for the top 25% of residues in
2v5wB, that are at the interface with zinc ion.
Fig. 9. Residues in 2v5wB, at the interface with zinc ion, colored by their
relative importance. The ligand (zinc ion) is colored green. Atoms further
than 30Å away from the geometric center of the ligand, as well as on the line
of sight to the ligand were removed. (See Appendix for the coloring scheme
for the protein chain 2v5wB.)
272
type
D
0.11
noc/
bb
6/2
dist
(Å)
3.73
0.14
21/2
3.20
0.16
1/0
4.56
0.16
11/4
3.30
0.17
20/13
3.21
0.19
3/2
4.72
antn
site
Table 12. The top 25% of residues in 2v5wB at the interface with 2v5wA.
(Field names: res: residue number in the PDB entry; type: amino acid type;
substs: substitutions seen in the alignment; with the percentage of each type
in the bracket; noc/bb: number of contacts with the ligand, with the number of
contacts realized through backbone atoms given in the bracket; dist: distance
of closest apporach to the ligand. )
Figure 9 shows residues in 2v5wB colored by their importance, at the
interface with 2v5wBZN1379.
Interface with 2v5wA.Table 12 lists the top 25% of residues at
the interface with 2v5wA. The following table (Table 13) suggests
possible disruptive replacements for these residues (see Section 3.6).
res
cvg
Table 12.
subst’s
cvg noc/
dist antn
(%)
bb
(Å)
D(93)
0.08 1/1 4.40
.(3)SPN
KQVE
continued in next column
8
res
type
272
89
275
202
D
D
C
K
Table 13.
disruptive
mutations
(R)(H)(FW)(Y)
(R)(FWH)(K)(Y)
(R)(E)(K)(FWH)
(Y)(T)(FW)(SCG)
continued in next column
Table 13. continued
res type disruptive
mutations
207
F
(E)(K)(T)(D)
F
(E)(K)(T)(D)
306
P
(R)(Y)(H)(T)
273
Table 14. continued
res type subst’s
(%)
176
D
D(97)TH
.EN
181
H
H(94)A
Q(1)
Y(1)F.R
W
142
H
H(96)L
.(1)SRP
Y
182
G
G(92)R
C(2)
S(1)
P(1)A.
179
L
I(29)
V(39)Y
A(13)
L(10)
C(1)
M(2).GS
FPH
Table 13. List of disruptive mutations for the top 25% of residues in
2v5wB, that are at the interface with 2v5wA.
cvg
0.02
noc/
bb
7/3
dist
(Å)
2.65
0.02
3/3
4.40
0.05
1/0
4.83
0.08
1/1
4.33
0.15
3/3
4.65
antn
site
Table 14. The top 25% of residues in 2v5wB at the interface with potassium ion.(Field names: res: residue number in the PDB entry; type: amino
acid type; substs: substitutions seen in the alignment; with the percentage of
each type in the bracket; noc/bb: number of contacts with the ligand, with
the number of contacts realized through backbone atoms given in the bracket;
dist: distance of closest apporach to the ligand. )
Fig. 10. Residues in 2v5wB, at the interface with 2v5wA, colored by
their relative importance. 2v5wA is shown in backbone representation (See
Appendix for the coloring scheme for the protein chain 2v5wB.)
Figure 10 shows residues in 2v5wB colored by their importance, at
the interface with 2v5wA.
Potassium ion binding site. Table 14 lists the top 25% of residues
at the interface with 2v5wBK1377 (potassium ion). The following
table (Table 15) suggests possible disruptive replacements for these
residues (see Section 3.6).
res
type
178
D
180
H
199
S
201
H
res
type
178
180
199
201
176
181
142
182
179
D
H
S
H
D
H
H
G
L
Table 15.
disruptive
mutations
(R)(FWH)(K)(QM)
(E)(Q)(K)(M)
(KR)(Q)(H)(M)
(E)(Q)(D)(K)
(R)(FW)(H)(VA)
(E)(D)(T)(Q)
(E)(T)(D)(Q)
(E)(KR)(H)(D)
(R)(Y)(K)(H)
Table 15. List of disruptive mutations for the top 25% of residues in
2v5wB, that are at the interface with potassium ion.
Table 14.
subst’s
cvg noc/
dist antn
(%)
bb
(Å)
D(97)S. 0.00 5/4 2.76 site
TG
H(97)SL 0.01 4/4 2.77 site
.TPD
S(97)V. 0.01 4/2 2.96
FDT
H(97)F. 0.01 5/2 3.83
TPV
continued in next column
Figure 11 shows residues in 2v5wB colored by their importance, at
the interface with 2v5wBK1377.
Interface with the peptide 2v5wI. Table 16 lists the top 25%
of residues at the interface with 2v5wI. The following table (Table
17) suggests possible disruptive replacements for these residues (see
Section 3.6).
9
Fig. 11. Residues in 2v5wB, at the interface with potassium ion, colored by
their relative importance. The ligand (potassium ion) is colored green. Atoms
further than 30Å away from the geometric center of the ligand, as well as on
the line of sight to the ligand were removed. (See Appendix for the coloring
scheme for the protein chain 2v5wB.)
res
type
306
F
273
P
subst’s
(%)
Y(91)
.(5)KLF
NHW
R(61)
L(1)
K(6)
.(3)YT
P(22)VE
HDCMG
Table 16.
cvg
0.17
0.19
noc/
bb
5/5
30/12
dist
(Å)
3.48
3.54
Fig. 12. Residues in 2v5wB, at the interface with 2v5wI, colored by their relative importance. 2v5wI is shown in backbone representation (See Appendix
for the coloring scheme for the protein chain 2v5wB.)
Figure 12 shows residues in 2v5wB colored by their importance, at
the interface with 2v5wI.
MCM binding site. Table 18 lists the top 25% of residues at
the interface with 2v5wIMCM6 (mcm). The following table (Table
19) suggests possible disruptive replacements for these residues (see
Section 3.6).
antn
site
Table 16. The top 25% of residues in 2v5wB at the interface with 2v5wI.
(Field names: res: residue number in the PDB entry; type: amino acid type;
substs: substitutions seen in the alignment; with the percentage of each type
in the bracket; noc/bb: number of contacts with the ligand, with the number of
contacts realized through backbone atoms given in the bracket; dist: distance
of closest apporach to the ligand. )
res
306
273
Table 17.
type disruptive
mutations
F
(E)(K)(T)(D)
P
(R)(Y)(H)(T)
Table 17. List of disruptive mutations for the top 25% of residues in
2v5wB, that are at the interface with 2v5wI.
10
res
type
152
F
306
F
273
P
274
M
Table 18.
subst’s
cvg
noc/
dist antn
(%)
bb
(Å)
F(96)
0.07
4/0 3.78 site
Y(1)H.S
LA
Y(91)
0.17
8/0 4.48
.(5)KLF
NHW
R(61)
0.19 11/2 3.50 site
L(1)
K(6)
.(3)YT
P(22)VE
HDCMG
I(3)
0.20
1/0 4.06
L(80)
.(3)
H(1)V
F(2)
Y(1)
Q(1)
continued in next column
Table 18. continued
res type subst’s
(%)
M(3)ASC
cvg
noc/
bb
dist
(Å)
susbtantially larger than) other functional sites and interfaces recognizable in PDB entry 2v5w. It is shown in Fig. 14. The right panel
shows (in blue) the rest of the larger cluster this surface belongs to.
antn
Table 18. The top 25% of residues in 2v5wB at the interface with
MCM.(Field names: res: residue number in the PDB entry; type: amino acid
type; substs: substitutions seen in the alignment; with the percentage of each
type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist:
distance of closest apporach to the ligand. )
res
type
152
306
273
274
F
F
P
M
Table 19.
disruptive
mutations
(K)(E)(Q)(D)
(E)(K)(T)(D)
(R)(Y)(H)(T)
(Y)(H)(TR)(SCG)
Fig. 14. A possible active surface on the chain 2v5wB. The larger cluster it
belongs to is shown in blue.
The residues belonging to this surface ”patch” are listed in Table 20,
while Table 21 suggests possible disruptive replacements for these
residues (see Section 3.6).
Table 19. List of disruptive mutations for the top 25% of residues in
2v5wB, that are at the interface with MCM.
Fig. 13. Residues in 2v5wB, at the interface with MCM, colored by their
relative importance. The ligand (MCM) is colored green. Atoms further than
30Å away from the geometric center of the ligand, as well as on the line of
sight to the ligand were removed. (See Appendix for the coloring scheme for
the protein chain 2v5wB.)
Figure 13 shows residues in 2v5wB colored by their importance, at
the interface with 2v5wIMCM6.
2.4.3 Possible novel functional surfaces at 25% coverage. One
group of residues is conserved on the 2v5wB surface, away from (or
11
res
180
199
183
151
265
71
142
209
143
189
237
152
267
182
type
H
S
D
G
G
H
H
P
H
F
D
F
D
G
272
D
103
P
186
E
174
Y
101
D
197
T
105
T
Table 20.
substitutions(%)
cvg antn
H(97)SL.TPD
0.01 site
S(97)V.FDT
0.01
D(95)TN(2)Q.G
0.02
G(98)R.WS
0.03
G(96).(3)AR
0.03
H(94).(4)VYTE
0.05
H(96)L.(1)SRPY
0.05
P(96)F.(1)KSRQ
0.05
H(96)QA.(1)NS
0.06
F(96)YL(1).MAH
0.06 site
D(94).(2)ARGEN
0.06
F(96)Y(1)H.SLA
0.07 site
D(94).(3)HNGSFT 0.07 site
G(92)RC(2)S(1)
0.08
P(1)A.
D(93).(3)SPNKQV 0.08
E
P(91)Y(1).(4)
0.09
A(1)ENSMR
E(66)CQ(25)A(2) 0.09
S(1)D(1).KFP
Y(92)EI(2)V(2)A 0.10
.LW
D(86)G(2)E(3)
0.11 site
.(4)TS(2)PKN
T(88)GV(1)I(1)S 0.11
Y(2)M(1)L(1).QC
K
F(84)S(1)V(2)
0.12
continued in next column
Table 20. continued
res type substitutions(%)
cvg antn
.(3)L(1)AIYT(2)
WM(1)NRH
208
F
F(88)IPY(3).(1) 0.12
W(4)LGX
218
G
G(89)E.(2)KPRSV 0.13
A(2)DQTL
154
Y
Y(84)F(3)I(4)
0.14
V(4)HR.L(1)D
275
C
G(72).(3)S(4)
0.14
K(2)T(4)A(6)
N(1)C(2)RV(1)EI
H
277
F
F(65)L(20).(3)
0.14
M(1)W(3)Y(2)TVC
I
304
G
G(92).(4)YERKSD 0.14
N
188
A
A(87)F(1)I(2)
0.16
G(2)L(1)V(1)TYM
.PSX
202
K
K(63)Q(6)G(2)
0.16
R(6)CL(1)H(2)
E(12).(1)A(1)
M(1)PNFS
207
F
F(52)Y(29)L(9)N 0.16
I(1).(1)R(2)APV
W
306
F
Y(91).(5)KLFNHW 0.17
37
R
R(89)K(1).(6)HP 0.18
VEFT
141
W
L(70)M(6)T(4)
0.18
R(5)W(5)S.(2)K
F(1)G(1)YN
210
G
G(85)R(1)E(1)P
0.18
C(1)A(1).(1)LY
F(2)K(1)NQSDV
315
W
W(83).(11)HY(1) 0.18
ILTA(1)SF
195
V
V(92)RI(2)LT(1) 0.19
C(1).FA
211
T
T(87)IS(7)KP
0.19
.(1)RHEVQYN
273
P
R(61)L(1)K(6)
0.19 site
.(3)YTP(22)VEHD
CMG
137
W
W(74)T(1)P(5)
0.20
Y(5).(1)VL(5)
I(2)MRF(1)HE
266
A
A(66).(3)V(3)
0.20
T(3)C(4)G(11)
F(2)S(4)MPEL
274
M
I(3)L(80).(3)
0.20
continued in next column
Table 20. continued
res type substitutions(%)
H(1)VF(2)Y(1)
Q(1)M(3)ASC
192
T
T(62)N(2)VS(15)
D(14)PCR(1)E.LF
216
D
D(61)T(1)E(25)
L(1)Y.(2)S(3)
N(1)AMQ
313
R
R(81).(7)S(2)Y
K(5)QPLDAHF
280
T
S(52)T(34).(4)W
N(6)IKHLQR
98
L
L(25)QV(37)G(3)
.(7)I(13)E(2)
F(6)MD(1)RANC
150
S
S(80)T(1)N(3)
A(5)G(3)EYF.HRM
DCV
269
I
L(75)I(3).(3)
H(8)M(1)F(2)
V(4)Y(1)D
70
F
F(70)Y(12)V(7)
.(4)W(2)I(2)ASC
111
Y
F(46)L(1)Y(29)
R(5)G.(3)A(6)
W(2)VH(1)SCT
97
G
N(54)S(2)G(27)
.(6)L(2)D(1)FAH
IKTQC
160
L
L(76)I(13)V(7).
AQTMC
cvg
antn
0.21
site
0.21
0.21
0.22
0.23
0.23
0.23
0.24
0.24
0.25
0.25
Table 20. Residues forming surface ”patch” in 2v5wB.
12
res
type
180
199
183
151
265
71
142
209
143
189
237
152
267
182
272
103
H
S
D
G
G
H
H
P
H
F
D
F
D
G
D
P
Table 21.
disruptive
mutations
(E)(Q)(K)(M)
(KR)(Q)(H)(M)
(R)(FWH)(Y)(K)
(E)(K)(D)(R)
(E)(KD)(R)(FQMWH)
(E)(Q)(M)(K)
(E)(T)(D)(Q)
(Y)(T)(R)(H)
(E)(TD)(M)(Q)
(K)(E)(T)(Q)
(R)(FWH)(Y)(VCAG)
(K)(E)(Q)(D)
(R)(FKWH)(Y)(VQMA)
(E)(KR)(H)(D)
(R)(H)(FW)(Y)
(Y)(R)(H)(T)
continued in next column
Table 21. continued
res type disruptive
mutations
186
E
(H)(FW)(Y)(R)
Y
(K)(QR)(E)(M)
174
D
(R)(FWH)(Y)(K)
101
197
T
(R)(K)(H)(FW)
T
(K)(R)(Q)(E)
105
F
(K)(E)(Q)(R)
208
G
(R)(H)(K)(E)
218
154
Y
(K)(Q)(M)(E)
C
(R)(E)(K)(FWH)
275
F
(K)(E)(Q)(R)
277
304
G
(R)(FW)(K)(E)
A
(R)(K)(E)(Y)
188
K
(Y)(T)(FW)(SCG)
202
207
F
(E)(K)(T)(D)
F
(E)(K)(T)(D)
306
R
(T)(D)(Y)(E)
37
W
(E)(K)(D)(T)
141
210
G
(R)(KE)(H)(FW)
W
(K)(E)(Q)(D)
315
V
(E)(KR)(Y)(D)
195
211
T
(R)(K)(FWH)(M)
P
(R)(Y)(H)(T)
273
W
(K)(E)(T)(Q)
137
A
(R)(K)(Y)(E)
266
274
M
(Y)(H)(TR)(SCG)
T
(R)(K)(H)(FW)
192
D
(R)(H)(FW)(Y)
216
313
R
(T)(D)(Y)(E)
T
(R)(K)(FWH)(EM)
280
L
(Y)(R)(H)(T)
98
150
S
(KR)(QH)(FMW)(E)
I
(R)(Y)(T)(K)
269
F
(K)(E)(Q)(D)
70
Y
(K)(Q)(E)(M)
111
97
G
(R)(E)(K)(H)
L
(Y)(R)(H)(T)
160
3.2
One of the table columns is “substitutions” - other amino acid types
seen at the same position in the alignment. These amino acid types
may be interchangeable at that position in the protein, so if one wants
to affect the protein by a point mutation, they should be avoided. For
example if the substitutions are “RVK” and the original protein has
an R at that position, it is advisable to try anything, but RVK. Conversely, when looking for substitutions which will not affect the protein,
one may try replacing, R with K, or (perhaps more surprisingly), with
V. The percentage of times the substitution appears in the alignment
is given in the immediately following bracket. No percentage is given
in the cases when it is smaller than 1%. This is meant to be a rough
guide - due to rounding errors these percentages often do not add up
to 100%.
3.3
3.4
Number of contacts
Another column worth noting is denoted “noc/bb”; it tells the number of contacts heavy atoms of the residue in question make across
the interface, as well as how many of them are realized through the
backbone atoms (if all or most contacts are through the backbone,
mutation presumably won’t have strong impact). Two heavy atoms
are considered to be “in contact” if their centers are closer than 5Å.
3.5
Annotation
If the residue annotation is available (either from the pdb file or
from other sources), another column, with the header “annotation”
appears. Annotations carried over from PDB are the following: site
(indicating existence of related site record in PDB ), S-S (disulfide
bond forming residue), hb (hydrogen bond forming residue, jb (james
bond forming residue), and sb (for salt bridge forming residue).
3.6
3.1
Surface
To detect candidates for novel functional interfaces, first we look for
residues that are solvent accessible (according to DSSP program) by
at least 10Å2 , which is roughly the area needed for one water molecule to come in the contact with the residue. Furthermore, we require
that these residues form a “cluster” of residues which have neighbor
within 5Å from any of their heavy atoms.
Note, however, that, if our picture of protein evolution is correct,
the neighboring residues which are not surface accessible might be
equally important in maintaining the interaction specificity - they
should not be automatically dropped from consideration when choosing the set for mutagenesis. (Especially if they form a cluster with
the surface residues.)
Table 21. Disruptive mutations for the surface patch in 2v5wB.
3
Known substitutions
Mutation suggestions
Mutation suggestions are completely heuristic and based on complementarity with the substitutions found in the alignment. Note that
they are meant to be disruptive to the interaction of the protein
with its ligand. The attempt is made to complement the following
properties: small [AV GST C], medium [LP N QDEM IK], large
[W F Y HR], hydrophobic [LP V AM W F I], polar [GT CY ]; positively [KHR], or negatively [DE] charged, aromatic [W F Y H],
long aliphatic chain [EKRQM ], OH-group possession [SDET Y ],
and NH2 group possession [N QRK]. The suggestions are listed
according to how different they appear to be from the original amino
acid, and they are grouped in round brackets if they appear equally
disruptive. From left to right, each bracketed group of amino acid
types resembles more strongly the original (i.e. is, presumably, less
NOTES ON USING TRACE RESULTS
Coverage
Trace results are commonly expressed in terms of coverage: the residue is important if its “coverage” is small - that is if it belongs to
some small top percentage of residues [100% is all of the residues
in a chain], according to trace. The ET results are presented in the
form of a table, usually limited to top 25% percent of residues (or
to some nearby percentage), sorted by the strength of the presumed
evolutionary pressure. (I.e., the smaller the coverage, the stronger the
pressure on the residue.) Starting from the top of that list, mutating a
couple of residues should affect the protein somehow, with the exact
effects to be determined experimentally.
13
4.3
4.3.1 Alistat alistat reads a multiple sequence alignment from the
file and shows a number of simple statistics about it. These statistics include the format, the number of sequences, the total number
of residues, the average and range of the sequence lengths, and the
alignment length (e.g. including gap characters). Also shown are
some percent identities. A percent pairwise alignment identity is defined as (idents / MIN(len1, len2)) where idents is the number of
exact identities and len1, len2 are the unaligned lengths of the two
sequences. The ”average percent identity”, ”most related pair”, and
”most unrelated pair” of the alignment are the average, maximum,
and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant
seq” is calculated by finding the maximum pairwise identity (best
relative) for all N sequences, then finding the minimum of these N
numbers (hence, the most outlying sequence). alistat is copyrighted
by HHMI/Washington University School of Medicine, 1992-2001,
and freely distributed under the GNU General Public License.
COVERAGE
V
50%
30%
5%
V
100%
RELATIVE IMPORTANCE
Fig. 15. Coloring scheme used to color residues by their relative importance.
4.3.2 CE To map ligand binding sites from different
source structures,
report maker uses the CE program:
http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998)
”Protein structure alignment by incremental combinatorial extension
(CE) of the optimal path . Protein Engineering 11(9) 739-747.
disruptive) These suggestions are tentative - they might prove disruptive to the fold rather than to the interaction. Many researcher will
choose, however, the straightforward alanine mutations, especially in
the beginning stages of their investigation.
4
4.3.3 DSSP In this work a residue is considered solvent accessible if the DSSP program finds it exposed to water by at least 10Å2 ,
which is roughly the area needed for one water molecule to come in
the contact with the residue. DSSP is copyrighted by W. Kabsch, C.
Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version
by Elmar.Krieger@cmbi.kun.nl November 18,2002,
APPENDIX
4.1
Credits
File formats
Files with extension “ranks sorted” are the actual trace results. The
fields in the table in this file:
• alignment# number of the position in the alignment
http://www.cmbi.kun.nl/gv/dssp/descrip.html.
• residue# residue number in the PDB file
4.3.4 HSSP Whenever available, report maker uses HSSP alignment as a starting point for the analysis (sequences shorter than
75% of the query are taken out, however); R. Schneider, A. de
Daruvar, and C. Sander. ”The HSSP database of protein structuresequence alignments.” Nucleic Acids Res., 25:226–230, 1997.
• type amino acid type
• rank rank of the position according to older version of ET
• variability has two subfields:
1. number of different amino acids appearing in in this column
of the alignment
http://swift.cmbi.kun.nl/swift/hssp/
2. their type
• rho ET score - the smaller this value, the lesser variability of
this position across the branches of the tree (and, presumably,
the greater the importance for the protein)
4.3.5 LaTex The text for this report was processed using LATEX;
Leslie Lamport, “LaTeX: A Document Preparation System AddisonWesley,” Reading, Mass. (1986).
• cvg coverage - percentage of the residues on the structure which
have this rho or smaller
4.3.6 Muscle When making alignments “from scratch”, report
maker uses Muscle alignment program: Edgar, Robert C. (2004),
”MUSCLE: multiple sequence alignment with high accuracy and
high throughput.” Nucleic Acids Research 32(5), 1792-97.
• gaps percentage of gaps in this column
4.2
Color schemes used
The following color scheme is used in figures with residues colored
by cluster size: black is a single-residue cluster; clusters composed of
more than one residue colored according to this hierarchy (ordered
by descending size): red, blue, yellow, green, purple, azure, turquoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold,
bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine,
DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen,
tan, DarkOrange, DeepPink, maroon, BlanchedAlmond.
The colors used to distinguish the residues by the estimated
evolutionary pressure they experience can be seen in Fig. 15.
http://www.drive5.com/muscle/
4.3.7 Pymol The figures in this report were produced using
Pymol. The scripts can be found in the attachment. Pymol
is an open-source application copyrighted by DeLano Scientific LLC (2005). For more information about Pymol see
http://pymol.sourceforge.net/. (Note for Windows
users: the attached package needs to be unzipped for Pymol to read
the scripts and launch the viewer.)
14
4.4
Note about ET Viewer
• 2v5wB.complex.pdb - coordinates of 2v5wB with all of its
interacting partners
Dan Morgan from the Lichtarge lab has developed a visualization
tool specifically for viewing trace results. If you are interested, please
visit:
• 2v5wB.etvx - ET viewer input file for 2v5wB
• 2v5wB.cluster report.summary - Cluster report summary for
2v5wB
http://mammoth.bcm.tmc.edu/traceview/
• 2v5wB.ranks - Ranks file in sequence order for 2v5wB
The viewer is self-unpacking and self-installing. Input files to be used
with ETV (extension .etvx) can be found in the attachment to the
main report.
4.5
• 2v5wB.clusters - Cluster descriptions for 2v5wB
• 2v5wB.msf - the multiple sequence alignment used for the chain
2v5wB
Citing this work
• 2v5wB.descr - description of sequences used in 2v5wB msf
The method used to rank residues and make predictions in this report
can be found in Mihalek, I., I. Reš, O. Lichtarge. (2004). ”A Family of
Evolution-Entropy Hybrid Methods for Ranking of Protein Residues
by Importance” J. Mol. Bio. 336: 1265-82. For the original version
of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolutionary Trace Method Defines Binding Surfaces Common to Protein
Families” J. Mol. Bio. 257: 342-358.
report maker itself is described in Mihalek I., I. Res and O.
Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type
of service for comparative analysis of proteins.” Bioinformatics
22:1656-7.
4.6
• 2v5wB.ranks sorted - full listing of residues and their ranking
for 2v5wB
• 2v5wB.2v5wL.if.pml - Pymol script for Figure 5
• 2v5wB.cbcvg - used by other 2v5wB – related pymol scripts
• 2v5wB.2v5wG.if.pml - Pymol script for Figure 6
• 2v5wB.2v5wBK1378.if.pml - Pymol script for Figure 7
• 2v5wB.2v5wLMCM6.if.pml - Pymol script for Figure 8
• 2v5wB.2v5wBZN1379.if.pml - Pymol script for Figure 9
• 2v5wB.2v5wA.if.pml - Pymol script for Figure 10
About report maker
• 2v5wB.2v5wBK1377.if.pml - Pymol script for Figure 11
report maker was written in 2006 by Ivana Mihalek. The 1D ranking visualization program was written by Ivica Reš. report maker
is copyrighted by Lichtarge Lab, Baylor College of Medicine,
Houston.
4.7
• 2v5wB.2v5wI.if.pml - Pymol script for Figure 12
• 2v5wB.2v5wIMCM6.if.pml - Pymol script for Figure 13
Attachments
The following files should accompany this report:
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