2xav Lichtarge lab 2006

Pages 1–11
2xav
Evolutionary trace report by report maker
July 16, 2010
4.4
4.5
4.6
4.7
1
CONTENTS
1
Introduction
1
2
Chain 2xavA
2.1 P00452 overview
2.2 Multiple sequence alignment for 2xavA
2.3 Residue ranking in 2xavA
2.4 Top ranking residues in 2xavA 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
2
2
3
5
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
9
9
9
9
10
10
10
4
Appendix
4.1 File formats
4.2 Color schemes used
4.3 Credits
10
10
10
10
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
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INTRODUCTION
From the original Protein Data Bank entry (PDB id 2xav):
Title: Ribonucleotide reductase y731no2y and y730f modified r1
subunit of e. coli
Compound: Mol id: 1; molecule: ribonucleoside-diphosphate reductase 1 subunit alpha; chain: a, b, c; fragment: residues 1-761;
synonym: ribonucleoside-diphosphate reductase 1 r1 subunit, ribonucleotide reductase 1, protein b1, ribonucleotide reductase r1 subunit; ec: 1.17.4.1; engineered: yes; mutation: yes; mol id: 2; molecule:
ribonucleoside-diphosphate reductase 1 subunit beta; chain: d, e,
f, p; fragment: ribonucleotide reductase r2-peptide, residues 357376; synonym: ribonucleotide reductase 1, protein b2, protein r2;
ec: 1.17.4.1
Organism, scientific name: Unidentified;
2xav contains a single unique chain 2xavA (728 residues long)
and its homologues 2xavC and 2xavB. Chains 2xavD, 2xavE, 2xavF,
2xavP, 2xavF1, 2xavF2, 2xavF3, 2xavF4, and 2xavF5 are too short to
permit statistically significant analysis, and were treated as a peptide
ligands.
2 CHAIN 2XAVA
2.1 P00452 overview
From SwissProt, id P00452, 98% identical to 2xavA:
Description: Ribonucleoside-diphosphate reductase 1 alpha subunit
(EC 1.17.4.1) (Ribonucleoside-diphosphate reductase 1 R1 subunit)
(Ribonucleotide reductase 1) (B1 protein).
Organism, scientific name: Escherichia coli.
Taxonomy: Bacteria; Proteobacteria; Gammaproteobacteria;
Enterobacteriales; Enterobacteriaceae; Escherichia.
Function: Provides the precursors necessary for DNA synthesis.
Catalyzes the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides. R1 contains the binding sites for both
substrates and allosteric effectors and carries out the actual reduction
of the ribonucleotide. It also provides redox- active cysteines.
Catalytic activity: 2’-deoxyribonucleoside diphosphate + thioredoxin disulfide + H(2)O = ribonucleoside diphosphate + thioredoxin.
1
Lichtarge lab 2006
Enzyme regulation: Allosterically regulated by nucleoside triphosphates. Stimulated by ATP and inhibited by dATP. It seems probable
that ATP makes the enzyme reduce CDP and UDP, dGTP favors ADP
reduction and dTTP favors GDP reduction. In vitro, its activity is
increased by dithiothreitol (DTT) or thioredoxins (non-specific).
Pathway: DNA replication pathway; first step.
Subunit: Tetramer of two alpha (R1) and two beta (R2) subunits. The
B1 protein is a dimer of alpha subunits. A radical transfer pathway
occurs between Tyr-122 of R2 and R1.
Interaction:
Alternative products:
Event=Alternative initiation; Comment=2 isoforms, Alpha (shown
here) and Alpha’, are produced by alternative initiation;
Ptm: Binding of the substrate occurs primarily when the active- site
cysteines are reduced.
Miscellaneous: E.coli produces two separate class I enzymes. This
one is the functional enzyme during growth.
Miscellaneous: Two distinct regulatory sites have been defined: one
controls substrate specificity and the other regulates the overall catalytic activity. A substrate-binding catalytic site, located on R1, is
formed only in the presence of the second subunit R2.
Similarity: Belongs to the ribonucleoside diphosphate reductase
large chain family.
Caution: Ref.2 sequence was incorrect.
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 4-245 in 2xavA colored by their relative importance. (See
Appendix, Fig.14, for the coloring scheme.)
Fig. 2. Residues 246-493 in 2xavA colored by their relative importance. (See
Appendix, Fig.14, for the coloring scheme.)
Multiple sequence alignment for 2xavA
For the chain 2xavA, the alignment 2xavA.msf (attached) with 186
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 2xavA.msf. Its statistics, from the
alistat program are the following:
Format:
MSF
Number of sequences: 186
Total number of residues:
Smallest:
667
Largest:
728
Average length:
719.6
Alignment length:
728
Average identity:
60%
Most related pair:
99%
Most unrelated pair: 21%
Most distant seq:
38%
133839
Fig. 3. Residues 494-737 in 2xavA colored by their relative importance. (See
Appendix, Fig.14, for the coloring scheme.)
Furthermore, 4% of residues show as conserved in this alignment.
The alignment consists of 2% eukaryotic ( <1% arthropoda, <1%
fungi, 1% plantae), 9% prokaryotic, and 5% viral sequences. (Descriptions of some sequences were not readily available.) The file
containing the sequence descriptions can be found in the attachment,
under the name 2xavA.descr.
2.3
Residue ranking in 2xavA
The 2xavA sequence is shown in Figs. 1–3, with each residue colored
according to its estimated importance. The full listing of residues
in 2xavA can be found in the file called 2xavA.ranks sorted in the
attachment.
2
2.4
Top ranking residues in 2xavA and their position on
the structure
In the following we consider residues ranking among top 25% of
residues in the protein . Figure 4 shows residues in 2xavA colored
by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A
Pymol script for producing this figure can be found in the attachment.
Fig. 5. Residues in 2xavA, 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.
Table 1. continued
cluster
size
color
Fig. 4. Residues in 2xavA, colored by their relative importance. Clockwise:
front, back, top and bottom views.
2.4.1 Clustering of residues at 25% coverage. Fig. 5 shows the
top 25% of all residues, this time colored according to clusters they
belong to. The clusters in Fig.5 are composed of the residues listed
in Table 1.
cluster
color
red
size
155
Table 1.
member
residues
67,75,76,77,81,82,83,145,147
150,151,154,155,167,206,208
209,210,211,212,214,216,217
220,221,223,224,225,226,232
234,237,247,248,249,251,252
253,254,255,262,275,276,277
280,281,283,286,300,301,303
307,309,310,312,316,319,321
322,324,329,331,334,338,344
346,353,355,358,375,379,382
383,395,409,410,413,418,421
436,437,438,439,441,442,444
continued in next column
blue
yellow
green
purple
azure
turquoise
7
5
4
2
2
2
member
residues
446,460,461,462,463,464,465
466,469,489,490,493,494,496
498,499,503,511,513,514,516
519,521,524,531,536,545,548
551,555,559,563,571,599,602
610,612,613,614,615,618,620
621,622,623,624,625,626,628
630,633,636,637,639,645,647
650,685,690,691,693,694,696
722,725,727,730,731,733
7,9,10,12,55,58,91
139,140,170,197,201
294,295,296,298
180,181
579,583
736,737
Table 1. Clusters of top ranking residues in 2xavA.
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 2xavD. Table 2 lists the top 25% of
residues at the interface with 2xavD. The following table (Table
3) suggests possible disruptive replacements for these residues (see
Section 3.6).
3
res
type
344
Y
722
Y
Table 2.
subst’s
cvg
(%)
Y(76)
0.15
M(19)
A(1)V
L(1)F
Y(76)
0.17
W(19)S
V(1)QHN
noc/
bb
9/3
dist
(Å)
3.61
18/2
3.79
Table 2. The top 25% of residues in 2xavA at the interface with 2xavD.
(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
344
722
Y
Y
Table 3.
disruptive
mutations
(K)(QR)(E)(NM)
(K)(M)(EQ)(R)
Table 3. List of disruptive mutations for the top 25% of residues in
2xavA, that are at the interface with 2xavD.
Fig. 6. Residues in 2xavA, at the interface with 2xavD, colored by their relative importance. 2xavD is shown in backbone representation (See Appendix
for the coloring scheme for the protein chain 2xavA.)
Figure 6 shows residues in 2xavA colored by their importance, at the
interface with 2xavD.
4
Interface with 2xavB.Table 4 lists the top 25% of residues at the
interface with 2xavB. The following table (Table 5) suggests possible
disruptive replacements for these residues (see Section 3.6).
res
type
280
283
P
K
294
Q
276
T
234
L
249
S
295
281
G
F
Table 4.
subst’s
cvg
(%)
P(99)K 0.10
K(89)
0.15
R(8)
D(1)A
Q(94)
0.19
G(4)TK
T(80)
0.21
.(16)
S(1)
Q(1)H
N(1)
L(84)
0.23
I(13)
G(1)A
S(76)
0.23
A(4)
K(15)
Q(3)C
G(88)
0.23
N(3)
L(6)A
Q(1)
F(85)
0.24
M(9)V
Y(2)
W(2)
noc/
bb
23/8
9/7
dist
(Å)
3.62
4.01
4/4
4.60
31/2
3.24
11/0
3.50
5/0
3.60
Fig. 7. Residues in 2xavA, at the interface with 2xavB, colored by their relative importance. 2xavB is shown in backbone representation (See Appendix
for the coloring scheme for the protein chain 2xavA.)
14/14
20/1
2.83
Interface with 2xavC1.By analogy with 2xavC – 2xavC1 interface. Table 6 lists the top 25% of residues at the interface with
2xavC1. The following table (Table 7) suggests possible disruptive
replacements for these residues (see Section 3.6).
3.43
Table 4. The top 25% of residues in 2xavA at the interface with 2xavB.
(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
280
283
294
276
234
249
295
281
P
K
Q
T
L
S
G
F
Table 5.
disruptive
mutations
(Y)(T)(HR)(SCG)
(Y)(FTW)(CG)(SVA)
(Y)(FW)(H)(T)
(R)(K)(FW)(H)
(R)(Y)(H)(KE)
(R)(FWH)(Y)(K)
(R)(E)(H)(Y)
(K)(E)(QD)(T)
res
type
650
G
647
S
Table 6.
subst’s
cvg
(%)
G(98)A 0.14
N(1)
S(81)
0.23
N(6)
T(2)
V(7)
D(1)
noc/
bb
9/9
dist
(Å)
4.09
9/9
3.32
Table 6. The top 25% of residues in 2xavA at the interface with 2xavC1.
(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. )
Table 5. List of disruptive mutations for the top 25% of residues in
2xavA, that are at the interface with 2xavB.
Figure 7 shows residues in 2xavA colored by their importance, at the
interface with 2xavB.
res
type
650
647
G
S
Table 7.
disruptive
mutations
(E)(R)(K)(H)
(R)(K)(H)(FW)
Table 7. List of disruptive mutations for the top 25% of residues in
2xavA, that are at the interface with 2xavC1.
5
belonging to this surface ”patch” are listed in Table 8, while Table
9 suggests possible disruptive replacements for these residues (see
Section 3.6).
res
9
12
55
91
10
type
K
G
T
K
R
7
58
V
I
Table 8.
substitutions(%)
K(98).(1)
G(98).NR
T(97)S(2)D
K(94)H(5)
R(94).(1)S(3)
L(1)
V(95).(1)I(2)A
I(86)L(12)AT
cvg
0.08
0.10
0.10
0.12
0.15
0.16
0.20
Table 8. Residues forming surface ”patch” in 2xavA.
Fig. 8. Residues in 2xavA, at the interface with 2xavC1, colored by their relative importance. 2xavC1 is shown in backbone representation (See Appendix
for the coloring scheme for the protein chain 2xavA.)
Figure 8 shows residues in 2xavA colored by their importance, at the
interface with 2xavC1.
res
type
9
12
55
91
10
7
58
K
G
T
K
R
V
I
Table 9.
disruptive
mutations
(Y)(FTW)(SVCAG)(HD)
(E)(FWD)(KMHR)(Y)
(R)(K)(FWH)(QM)
(TY)(SFVCAWG)(D)(E)
(TD)(Y)(ECG)(SVLAPI)
(YR)(KE)(H)(QD)
(R)(Y)(H)(K)
Table 9. Disruptive mutations for the surface patch in 2xavA.
2.4.3 Possible novel functional surfaces at 25% coverage. One
group of residues is conserved on the 2xavA surface, away from (or
susbtantially larger than) other functional sites and interfaces recognizable in PDB entry 2xav. It is shown in Fig. 9. The residues
Another group of surface residues is shown in Fig.10. The right panel
shows (in blue) the rest of the larger cluster this surface belongs to.
Fig. 10. Another possible active surface on the chain 2xavA. The larger
cluster it belongs to is shown in blue.
The residues belonging to this surface ”patch” are listed in Table 10,
while Table 11 suggests possible disruptive replacements for these
residues (see Section 3.6).
res
75
Fig. 9. A possible active surface on the chain 2xavA.
6
type
P
Table 10.
substitutions(%)
cvg
P(97)W(1)AQ
0.09
continued in next column
Table 10. continued
res type substitutions(%)
83
R
R(96)H(1)K(1)
N(1)E
77
Y
Y(91)W(7)H(1)
D
D(85)N(10)S(1)
76
E(1)HA(1)
33
L
L(93)T(2)I(1)
.(1)VK(1)
Table 12. continued
res type substitutions(%)
322
N
N(97)TP(1)Q
331
R
R(90)H(8)DK
K
K(89)R(8)D(1)A
283
N
N(81)K(14)R(1)T
321
D(1)Q
329
R
R(97)K(1)Q
T
T(80).(16)S(1)
276
Q(1)HN(1)
286
Q
H(1)Q(75)E(2)
N(15)D(3)G(1)T
234
L
L(84)I(13)G(1)A
281
F
F(85)M(9)VY(2)
W(2)
cvg
0.18
0.19
0.23
0.25
Table 10. Residues forming surface ”patch” in 2xavA.
res
type
75
83
77
76
33
P
R
Y
D
L
Table 11.
disruptive
mutations
(Y)(R)(T)(H)
(T)(Y)(D)(VCAG)
(K)(Q)(EM)(N)
(R)(FWH)(Y)(K)
(Y)(R)(H)(T)
Another group of surface residues is shown in Fig.11. The right panel
shows (in blue) the rest of the larger cluster this surface belongs to.
0.22
0.23
0.24
res
type
232
262
312
324
275
358
280
322
331
283
321
329
276
286
234
281
D
R
E
G
H
P
P
N
R
K
N
R
T
Q
L
F
Table 13.
disruptive
mutations
(R)(FWH)(KYVCAG)(TQM)
(TD)(SVCLAPIG)(YE)(FMW)
(FWH)(Y)(VCAG)(R)
(KR)(E)(QH)(FMW)
(E)(M)(Q)(D)
(R)(TKY)(E)(H)
(Y)(T)(HR)(SCG)
(Y)(H)(FW)(TR)
(T)(YVCADG)(S)(ELPI)
(Y)(FTW)(CG)(SVA)
(Y)(FW)(H)(T)
(T)(Y)(D)(SVCAG)
(R)(K)(FW)(H)
(Y)(FW)(H)(T)
(R)(Y)(H)(KE)
(K)(E)(QD)(T)
Table 13. Disruptive mutations for the surface patch in 2xavA.
Fig. 11. Another possible active surface on the chain 2xavA. The larger
cluster it belongs to is shown in blue.
Another group of surface residues is shown in Fig.12. The right panel
shows (in blue) the rest of the larger cluster this surface belongs to.
The residues belonging to this surface ”patch” are listed in Table 12,
while Table 13 suggests possible disruptive replacements for these
residues (see Section 3.6).
type
D
R
E
G
H
P
P
0.17
0.21
Table 12. Residues forming surface ”patch” in 2xavA.
Table 11. Disruptive mutations for the surface patch in 2xavA.
res
232
262
312
324
275
358
280
cvg
0.11
0.12
0.15
0.16
Table 12.
substitutions(%)
cvg
D(100)
0.05
R(98).(1)
0.07
E(81)D(17)Q
0.07
G(97)S(1)AT
0.07
H(82).(16)S(1) 0.08
P(98)LYF
0.08
P(99)K
0.10
continued in next column
Fig. 12. Another possible active surface on the chain 2xavA. The larger
cluster it belongs to is shown in blue.
7
The residues belonging to this surface ”patch” are listed in Table 14,
while Table 15 suggests possible disruptive replacements for these
residues (see Section 3.6).
res
421
725
727
346
418
383
344
type
N
G
K
R
D
E
Y
722
Y
382
690
Y
D
Table 14.
substitutions(%)
N(100)
G(100)
K(100)
R(98)K(1)
D(98)GH
E(98)VIM
Y(76)M(19)A(1)V
L(1)F
Y(76)W(19)SV(1)
QHN
Y(93)L(2)A(3)MV
D(95)C(3)SG
while Table 17 suggests possible disruptive replacements for these
residues (see Section 3.6).
res
155
210
224
225
253
298
300
437
438
439
441
462
498
516
563
610
621
637
694
696
252
511
612
636
296
410
464
461
630
208
645
221
493
620
466
623
633
212
519
625
217
460
731
521
650
214
628
cvg
0.05
0.05
0.05
0.06
0.06
0.14
0.15
0.17
0.18
0.19
Table 14. Residues forming surface ”patch” in 2xavA.
res
type
421
725
727
346
418
383
344
722
382
690
N
G
K
R
D
E
Y
Y
Y
D
Table 15.
disruptive
mutations
(Y)(FTWH)(SEVCARG)(MD)
(KER)(FQMWHD)(NYLPI)(SVA)
(Y)(FTW)(SVCAG)(HD)
(T)(YD)(SVCAG)(FELWPI)
(R)(K)(FW)(H)
(H)(Y)(R)(FW)
(K)(QR)(E)(NM)
(K)(M)(EQ)(R)
(K)(R)(Q)(E)
(R)(FWH)(K)(Y)
Table 15. Disruptive mutations for the surface patch in 2xavA.
Another group of surface residues is shown in Fig.13. The right panel
shows (in blue) the rest of the larger cluster this surface belongs to.
Fig. 13. Another possible active surface on the chain 2xavA. The larger
cluster it belongs to is shown in blue.
The residues belonging to this surface ”patch” are listed in Table 16,
8
type
Y
P
S
C
G
R
G
N
L
C
E
C
Y
G
G
G
P
P
S
N
A
R
R
E
G
G
L
L
N
P
K
Q
L
M
A
E
N
M
N
S
T
A
X
A
G
G
I
Table 16.
substitutions(%)
cvg
Y(100)
0.05
P(100)
0.05
S(100)
0.05
C(100)
0.05
G(100)
0.05
R(100)
0.05
G(100)
0.05
N(100)
0.05
L(100)
0.05
C(100)
0.05
E(100)
0.05
C(100)
0.05
Y(100)
0.05
G(100)
0.05
G(100)
0.05
G(100)
0.05
P(100)
0.05
P(100)
0.05
S(100)
0.05
N(98)D(1)
0.05
A(82)G(17)
0.06
R(98)M(1)
0.06
R(98)A(1)
0.06
E(97)DY(1)
0.06
G(98)Q(1)
0.07
G(98)A(1)
0.07
L(98)I(1)
0.07
L(81)V(17)T(1)
0.08
N(80)G(19)
0.08
P(81)A(17)S(1)
0.09
K(82)E(6)R(10)
0.09
Q(97)G(2)N
0.10
L(77)I(22)M
0.10
M(91)A(8)
0.10
A(80)S(18)G(1)
0.11
E(82)S(6)A(10)G 0.11
N(82)Q(6)E(10)P 0.11
M(80)L(18)Y(1)
0.12
N(79)G(20)
0.12
S(82)I(7)T(10)
0.12
T(96)F(1)RS(1)
0.13
A(95)PS(1)G(2)
0.13
Y(89).(9)X
0.13
A(96)Q(2)SH
0.14
G(98)AN(1)
0.14
G(81)N(17)SA
0.15
I(85)V(5)L(6)
0.15
A(1)
continued in next column
Table 16. continued
res type substitutions(%)
639
R
R(81)Y(6)T(9)F
V(1)K
666
Y
Y(98)IF
K
K(82)R(8)L(1)
154
S(8)
209
T
T(92)S(7)
Q
Q(81)T(17)IH
150
I
I(79)T(17)N(1)V
211
PY
248
V
V(76)I(3)S(17)
T(1)
622
S
C(6)S(72)T(18)
G(1)N
223
S
S(93)A(6)
463
T
T(77)I(2)N(16)
V(1)S(1)F
469
L
L(95)A(1)V(2)I
N
T(2)N(79)R(8)
733
.(9)H
294
Q
Q(94)G(4)TK
T
T(97)MS(2)
446
524
L
L(88)F(10)M(1)
L
L(90)I(8)MV
180
S
S(78)N(3)M(6)
206
T(10)L(1)
251
R
R(80)G(8)A(8)
S(2)Q
409
T
T(97)S(2)
531
Y
Y(83)W(6)F(9)
A
A(96)S(3)
559
L
L(90)V(2)M(4)
151
F(1)I(1)
220
R
R(81)H(6)A(1)
P(8)TE(1)S
490
D
D(91)E(1)N(7)
494
D
D(95)E(2)L(1)NS
Q
Q(80)N(16)M(2)A
496
S
730
F
Y(89).(1)M(8)F
S
S(76)A(4)K(15)
249
Q(3)C
295
G
G(88)N(3)L(6)A
Q(1)
499
P
P(97)A(1)V
S
S(81)N(6)T(2)
647
V(7)D(1)
301
A
A(91)S(8)
S
S(77)A(18)G(3)M
465
624
T
S(26)T(73)A
cvg
0.15
0.15
0.16
0.16
0.17
0.17
0.17
0.17
0.18
0.18
0.18
0.18
0.19
0.19
0.19
0.20
0.20
0.20
0.21
0.21
0.21
0.22
0.22
0.22
0.22
0.22
0.22
0.23
0.23
0.23
0.23
0.24
0.24
0.24
Table 16. Residues forming surface ”patch” in 2xavA.
9
res
type
155
210
224
225
253
298
300
437
438
439
441
462
498
516
563
610
621
637
694
696
252
511
612
636
296
410
464
461
630
208
645
221
493
620
466
623
633
212
519
625
217
460
731
521
650
214
628
639
666
Y
P
S
C
G
R
G
N
L
C
E
C
Y
G
G
G
P
P
S
N
A
R
R
E
G
G
L
L
N
P
K
Q
L
M
A
E
N
M
N
S
T
A
X
A
G
G
I
R
Y
Table 17.
disruptive
mutations
(K)(QM)(NEVLAPIR)(D)
(YR)(TH)(SKECG)(FQWD)
(KR)(FQMWH)(NYELPI)(D)
(KER)(FQMWHD)(NYLPI)(SVA)
(KER)(FQMWHD)(NYLPI)(SVA)
(TD)(SYEVCLAPIG)(FMW)(N)
(KER)(FQMWHD)(NYLPI)(SVA)
(Y)(FTWH)(SEVCARG)(MD)
(YR)(TH)(SKECG)(FQWD)
(KER)(FQMWHD)(NYLPI)(SVA)
(FWH)(YVCARG)(T)(SNKLPI)
(KER)(FQMWHD)(NYLPI)(SVA)
(K)(QM)(NEVLAPIR)(D)
(KER)(FQMWHD)(NYLPI)(SVA)
(KER)(FQMWHD)(NYLPI)(SVA)
(KER)(FQMWHD)(NYLPI)(SVA)
(YR)(TH)(SKECG)(FQWD)
(YR)(TH)(SKECG)(FQWD)
(KR)(FQMWH)(NYELPI)(D)
(Y)(FWH)(TR)(VCAG)
(KER)(Y)(QHD)(N)
(T)(YD)(SCG)(EVA)
(D)(TYE)(SCLPIG)(FVMAW)
(FWHR)(VA)(CG)(Y)
(FEWHR)(KYD)(M)(QLPI)
(KER)(QHD)(FYMW)(N)
(YR)(TH)(SKECG)(FQWD)
(R)(Y)(H)(K)
(Y)(FWH)(ER)(T)
(R)(Y)(H)(K)
(Y)(FTW)(VCAG)(S)
(Y)(FWH)(T)(SVA)
(Y)(R)(TH)(CG)
(Y)(H)(TR)(SCDG)
(KR)(E)(Y)(QH)
(H)(FWR)(Y)(K)
(Y)(H)(FTW)(CRG)
(Y)(T)(HR)(CG)
(Y)(FWH)(ER)(T)
(R)(K)(H)(Q)
(K)(R)(QM)(E)
(R)(K)(YE)(H)
(K)(ER)(QM)(D)
(YE)(KR)(D)(QH)
(E)(R)(K)(H)
(R)(KE)(H)(FW)
(YR)(H)(TKE)(SQCDG)
(D)(E)(T)(LPI)
(K)(Q)(R)(E)
continued in next column
Table 17. continued
res type disruptive
mutations
154
K
(Y)(FTW)(CG)(SVA)
T
(KR)(FQMWH)(NELPI)(D)
209
Q
(Y)(FTWH)(SVCAG)(D)
150
211
I
(R)(Y)(H)(K)
V
(R)(K)(YE)(H)
248
S
(R)(K)(FWH)(M)
622
S
(KR)(QH)(FYEMW)(N)
223
463
T
(R)(K)(H)(Q)
L
(YR)(H)(TKE)(SQCDG)
469
N
(Y)(FTW)(E)(VAH)
733
294
Q
(Y)(FW)(H)(T)
T
(R)(K)(H)(FW)
446
L
(YR)(T)(H)(KECG)
524
180
L
(Y)(R)(H)(T)
S
(R)(KH)(FW)(Y)
206
R
(D)(Y)(E)(T)
251
T
(KR)(FQMWH)(NELPI)(D)
409
531
Y
(K)(Q)(E)(M)
A
(KR)(YE)(QH)(D)
559
L
(R)(Y)(T)(H)
151
220
R
(D)(T)(Y)(E)
D
(R)(FWH)(Y)(VCAG)
490
D
(R)(H)(FW)(Y)
494
Q
(Y)(H)(FW)(T)
496
730
F
(K)(E)(TQD)(SNCRG)
S
(R)(FWH)(Y)(K)
249
G
(R)(E)(H)(Y)
295
499
P
(YR)(H)(KE)(T)
S
(R)(K)(H)(FW)
647
A
(KR)(YE)(QH)(D)
301
465
S
(R)(K)(H)(FYQW)
T
(KR)(QH)(FMW)(E)
624
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
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.)
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).
Table 17. Disruptive mutations for the surface patch in 2xavA.
3 NOTES ON USING TRACE RESULTS
3.1 Coverage
3.6
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
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.
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.
3.2
Surface
Known substitutions
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
10
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%
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.
V
100%
RELATIVE IMPORTANCE
Fig. 14. Coloring scheme used to color residues by their relative importance.
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 [email protected] November 18,2002,
4 APPENDIX
4.1 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. 14.
4.3
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.)
4.4
Credits
Note about ET Viewer
Dan Morgan from the Lichtarge lab has developed a visualization
tool specifically for viewing trace results. If you are interested, please
visit:
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
http://mammoth.bcm.tmc.edu/traceview/
11
4.7
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
Attachments
The following files should accompany this report:
• 2xavA.complex.pdb - coordinates of 2xavA with all of its
interacting partners
Citing this work
• 2xavA.etvx - ET viewer input file for 2xavA
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.
• 2xavA.cluster report.summary - Cluster report summary for
2xavA
• 2xavA.ranks - Ranks file in sequence order for 2xavA
• 2xavA.clusters - Cluster descriptions for 2xavA
• 2xavA.msf - the multiple sequence alignment used for the chain
2xavA
• 2xavA.descr - description of sequences used in 2xavA msf
• 2xavA.ranks sorted - full listing of residues and their ranking
for 2xavA
• 2xavA.2xavD.if.pml - Pymol script for Figure 6
4.6
About report maker
• 2xavA.cbcvg - used by other 2xavA – related pymol scripts
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.
• 2xavA.2xavB.if.pml - Pymol script for Figure 7
• 2xavA.2xavC1.if.pml - Pymol script for Figure 8
12