Pages 1–6 1ako Evolutionary trace report by report maker November 5, 2010

4.3.3 DSSP 5 4.3.4 HSSP 5 4.3.5 LaTex 5 4.3.6 Muscle 5 4.3.7 Pymol 5 4.4 Note about ET Viewer 6 4.5 Citing this work 6 4.6 About report maker 6 4.7 Attachments 6

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1ako): Title: iii from escherichia coli Compound: Mol id: 1; molecule: exonuclease iii; chain: a; ec: 3.1.11.2; engineered: yes Organism, scientific name: Escherichia Coli; 1ako contains a single unique chain 1akoA (268 residues long).

2 CHAIN 1AKOA 2.1 P09030 overview CONTENTS From SwissProt, id P09030, 100% identical to 1akoA: Description: III (EC 3.1.11.2) (Exonuclease 1 Introduction 1 III) (EXO III) (AP VI). 2 Chain 1akoA 1 Organism, scientific name: Escherichia coli. 2.1 P09030 overview 1 Taxonomy: Bacteria; Proteobacteria; Gammaproteobacteria; 2.2 Multiple sequence alignment for 1akoA 1 Enterobacteriales; Enterobacteriaceae; Escherichia. 2.3 Residue ranking in 1akoA 1 Function: Major apurinic-apyrimidinic endonuclease of E.coli. It 2.4 Top ranking residues in 1akoA and their position on removes the damaged DNA at cytosines and guanines by cleaving the structure 2 on the 3’ side of the AP site by a beta-elimination reaction. It exhi- 2.4.1 Clustering of residues at 25% coverage. 2 bits 3’-5’-exonuclease, 3’-phosphomonoesterase, 3’-repair diesterase 2.4.2 Possible novel functional surfaces at 25% and H activities. coverage. 2 Catalytic activity: Exonucleolytic cleavage in the 3’- to 5’- direction to yield nucleoside 5’-phosphates. 3 Notes on using trace results 4 Subunit: Monomer. 3.1 Coverage 4 Interaction: 3.2 Known substitutions 4 Similarity: Belongs to the DNA repair AP/exoA family. 3.3 Surface 4 About: This Swiss-Prot entry is copyright. It is produced through a 3.4 Number of contacts 4 collaboration between the Swiss Institute of Bioinformatics and the 3.5 Annotation 4 EMBL outstation - the European Bioinformatics Institute. There are 3.6 Mutation suggestions 4 no restrictions on its use as long as its content is in no way modified and this statement is not removed. 4 Appendix 5 4.1 File formats 5 2.2 Multiple sequence alignment for 1akoA 4.2 Color schemes used 5 For the chain 1akoA, the alignment 1akoA.msf (attached) with 97 4.3 Credits 5 sequences was used. The alignment was assembled through combi- 4.3.1 Alistat 5 nation of BLAST searching on the UniProt database and alignment 4.3.2 CE 5 using Muscle program. It can be found in the attachment to this

1 Lichtarge lab 2006 Fig. 1. Residues 1-134 in 1akoA colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.)

Fig. 2. Residues 135-268 in 1akoA colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.)

Fig. 3. Residues in 1akoA, colored by their relative importance. Clockwise: front, back, top and bottom views. report, under the name of 1akoA.msf. Its statistics, from the alistat program are the following: 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Format: MSF top 25% of all residues, this time colored according to clusters they Number of sequences: 97 belong to. The clusters in Fig.4 are composed of the residues listed Total number of residues: 24602 Smallest: 201 Largest: 268 Average length: 253.6 Alignment length: 268 Average identity: 34% Most related pair: 99% Most unrelated pair: 20% Most distant seq: 48%

Furthermore, 2% of residues show as conserved in this alignment. The alignment consists of 1% eukaryotic ( 1% vertebrata), 95% prokaryotic, and 3% archaean sequences. The file containing the sequence descriptions can be found in the attachment, under the name 1akoA.descr. 2.3 Residue ranking in 1akoA The 1akoA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues in 1akoA can be found in the file called 1akoA.ranks sorted in the attachment. 2.4 Top ranking residues in 1akoA and their position on the structure In the following we consider residues ranking among top 25% of Fig. 4. Residues in 1akoA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for residues in the protein . Figure 3 shows residues in 1akoA colored the coloring scheme). Clockwise: front, back, top and bottom views. The by their importance: bright red and yellow indicate more conser- corresponding Pymol script is attached. ved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. in Table 1.

2 Table 1. cluster size member color residues red 63 5,6,7,8,9,10,12,14,18,33,34 36,42,43,58,62,63,64,65,66 88,90,107,109,111,112,113 122,125,128,150,151,153,154 155,156,160,161,178,183,184 197,200,210,211,212,213,214 215,220,225,226,227,228,229 230,251,256,257,258,259,260 261 blue 2 51,52

Table 1. Clusters of top ranking residues in 1akoA.

2.4.2 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1akoA surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1ako. It is shown in Fig. 5. The right panel shows (in blue) the rest of the larger cluster this surface belongs to.

Fig. 5. A possible active surface on the chain 1akoA. The larger cluster it belongs to is shown in blue.

The residues belonging to this surface ”patch” are listed in Table 2, while Table 3 suggests possible disruptive replacements for these residues (see Section 3.6). Table 2. res type substitutions(%) cvg antn 9 N N(98)D(1) 0.03 34 E E(100) 0.03 site 90 R R(100) 0.03 109 Y Y(100) 0.03 151 D D(100) 0.03 153 N N(100) 0.03 36 K K(92)R(7) 0.04 227 R R(98).(1) 0.06 229 D D(98).(1) 0.06 258 D D(98).(1) 0.06 259 H H(98).(1) 0.06 continued in next column

3 Table 2. continued Table 2. continued res type substitutions(%) cvg antn res type substitutions(%) cvg antn 111 P P(96)H(2)V(1) 0.08 161 I L(27)I(29)V(34) 0.23 212 W W(75)F(16)Y(8) 0.09 S(2)A(4)C(1) 251 R .(27)R(67)T(2) 0.09 M(1) S(3) 43 P S(5)P(79)Q(1) 0.24 261 P P(97)A(1).(1) 0.09 A(1)T(1)D(5) 112 Q S(9)N(60)K(1) 0.10 E(3)R(1)N(2) Q(16)R(2)A(10) .(1) 215 Y N(3)Y(92)W(1) 0.10 13 A S(6)T(15)A(56) 0.25 Q(1)S(2) V(4)K(4)L(10) 10 G G(60)S(36)N(3) 0.11 G(2)R(1) 197 D D(94).(2)E(2) 0.11 18 L G(25)V(19)L(42) 0.25 A(1) N(4)M(3)K(1) 113 G G(82)A(6)S(11) 0.12 A(1)I(2)D(1) 178 S G(34)M(4)L(18) 0.12 58 G Y(9)G(67)P(6) 0.25 H(9)C(7)S(26) S(7)E(2)A(2) 63 Y S(27)N(46)A(4) 0.13 D(3)N(1)K(1) Y(16)H(5) F(1) 128 F F(53)W(42)Y(4) 0.13 156 P H(30)L(3)P(61) 0.13 Table 2. Residues forming surface ”patch” in 1akoA. S(1)R(2)V(1) 184 R R(84)Q(5)L(2) 0.14 T(6)V(1)I(1) Table 3. 214 D S(44)D(50)N(3) 0.14 res type disruptive T(1)K(1) mutations 122 F Q(10)Y(17)F(48) 0.15 9 N (Y)(FWH)(TR)(VCAG) L(21)I(1)M(1) 34 E (FWH)(YVCARG)(T)(SNKLPI) 200 R R(92)M(1)K(1) 0.15 90 R (TD)(SYEVCLAPIG)(FMW)(N) L(1)Q(1)T(1) 109 Y (K)(QM)(NEVLAPIR)(D) P(1)D(1) 151 D (R)(FWH)(KYVCAG)(TQM) 12 R R(75)N(10)K(12) 0.16 153 N (Y)(FTWH)(SEVCARG)(MD) A(2) 36 K (Y)(T)(FW)(SVCAG) 14 R A(11)R(65)I(7) 0.16 227 R (TD)(SVCLAPIG)(YE)(FMW) H(1)C(3)W(1) 229 D (R)(FWH)(VCAG)(KY) V(10) 258 D (R)(FWH)(VCAG)(KY) 62 H Y(54)W(16)R(7) 0.17 259 H (E)(TQMD)(SNVCLAPIG)(K) H(15)F(6) 111 P (R)(Y)(T)(E) 228 I I(77)L(21).(1) 0.17 212 W (K)(E)(Q)(D) 210 F Y(63)W(5)F(29) 0.18 251 R (D)(LPI)(E)(FTYVMAW) L(1) 261 P (Y)(R)(H)(T) 230 L Y(31)H(37)F(7) 0.19 112 Q (Y)(FW)(TH)(CG) M(6)V(4)L(12) 215 Y (K)(M)(EQR)(VA) .(1) 10 G (R)(KE)(FWH)(M) 42 F L(15)F(69)I(8) 0.20 197 D (R)(H)(FW)(Y) T(3)M(1)V(3) 113 G (KR)(E)(QH)(FMW) 88 Q E(40)Q(43)S(2) 0.22 178 S (R)(K)(Q)(H) D(7)H(4)G(1) 63 Y (K)(Q)(M)(E) A(1).(1) 128 F (K)(E)(Q)(D) 220 F Y(2)W(29)F(46) 0.22 156 P (Y)(R)(T)(E) R(20).(1) 184 R (Y)(T)(D)(E) 256 P F(5)P(49)A(13) 0.22 214 D (R)(FW)(H)(Y) W(3)T(3)G(22) 122 F (KE)(T)(D)(R) S(2).(1) 200 R (TY)(D)(CG)(S) continued in next column continued in next column

4 Table 3. continued Note, however, that, if our picture of protein evolution is correct, res type disruptive the neighboring residues which are not surface accessible might be mutations equally important in maintaining the interaction specificity - they 12 R (TY)(D)(E)(SCG) should not be automatically dropped from consideration when choo- 14 R (D)(E)(T)(Y) sing the set for mutagenesis. (Especially if they form a cluster with 62 H (E)(D)(TQ)(M) the surface residues.) 228 I (Y)(R)(T)(H) 210 F (K)(E)(Q)(TD) 3.4 Number of contacts 230 L (R)(Y)(T)(K) Another column worth noting is denoted “noc/bb”; it tells the num- 42 F (K)(E)(R)(TQD) ber of contacts heavy atoms of the residue in question make across 88 Q (Y)(FWH)(T)(VCAG) the interface, as well as how many of them are realized through the 220 F (E)(K)(D)(T) backbone atoms (if all or most contacts are through the backbone, 256 P (R)(Y)(K)(EH) mutation presumably won’t have strong impact). Two heavy atoms 161 I (R)(Y)(H)(K) are considered to be “in contact” if their centers are closer than 5A˚ . 43 P (Y)(R)(H)(T) 13 A (Y)(E)(R)(K) 3.5 Annotation 18 L (Y)(R)(H)(T) 58 G (R)(K)(E)(H) 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 Table 3. Disruptive mutations for the surface patch in 1akoA. (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 NOTES ON USING TRACE RESULTS 3.6 Mutation suggestions 3.1 Coverage Mutation suggestions are completely heuristic and based on comple- Trace results are commonly expressed in terms of coverage: the resi- mentarity with the substitutions found in the alignment. Note that due is important if its “coverage” is small - that is if it belongs to they are meant to be disruptive to the interaction of the protein some small top percentage of residues [100% is all of the residues with its ligand. The attempt is made to complement the following in a chain], according to trace. The ET results are presented in the properties: small [AV GSTC], medium [LPNQDEMIK], large form of a table, usually limited to top 25% percent of residues (or [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- to some nearby percentage), sorted by the strength of the presumed tively [KHR], or negatively [DE] charged, aromatic [WFYH], evolutionary pressure. (I.e., the smaller the coverage, the stronger the long aliphatic chain [EKRQM], OH-group possession [SDETY ], pressure on the residue.) Starting from the top of that list, mutating a and NH2 group possession [NQRK]. The suggestions are listed couple of residues should affect the protein somehow, with the exact according to how different they appear to be from the original amino effects to be determined experimentally. acid, and they are grouped in round brackets if they appear equally disruptive. From left to right, each bracketed group of amino acid 3.2 Known substitutions types resembles more strongly the original (i.e. is, presumably, less One of the table columns is “substitutions” - other amino acid types disruptive) These suggestions are tentative - they might prove disrup- seen at the same position in the alignment. These amino acid types tive to the fold rather than to the interaction. Many researcher will may be interchangeable at that position in the protein, so if one wants choose, however, the straightforward alanine mutations, especially in to affect the protein by a point mutation, they should be avoided. For the beginning stages of their investigation. example if the substitutions are “RVK” and the original protein has an R at that position, it is advisable to try anything, but RVK. Conver- sely, when looking for substitutions which will not affect the protein, 4 APPENDIX one may try replacing, R with K, or (perhaps more surprisingly), with 4.1 File formats V. The percentage of times the substitution appears in the alignment Files with extension “ranks sorted” are the actual trace results. The is given in the immediately following bracket. No percentage is given fields in the table in this file: 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 • alignment# number of the position in the alignment to 100%. • residue# residue number in the PDB file 3.3 Surface • type amino acid type To detect candidates for novel functional interfaces, first we look for • rank rank of the position according to older version of ET residues that are solvent accessible (according to DSSP program) by 2 • variability has two subfields: at least 10A˚ , which is roughly the area needed for one water mole- 1. number of different amino acids appearing in in this column cule to come in the contact with the residue. Furthermore, we require of the alignment that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms. 2. their type

5 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. 4.3.3 DSSP In this work a residue is considered solvent accessi- COVERAGE ble if the DSSP program finds it exposed to water by at least 10A˚ 2, which is roughly the area needed for one water molecule to come in V the contact with the residue. DSSP is copyrighted by W. Kabsch, C. 100% 50% 30% 5% Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by [email protected] November 18,2002, http://www.cmbi.kun.nl/gv/dssp/descrip.html.

4.3.4 HSSP Whenever available, report maker uses HSSP ali-

V gnment as a starting point for the analysis (sequences shorter than RELATIVE IMPORTANCE 75% of the query are taken out, however); R. Schneider, A. de Daruvar, and C. Sander. ”The HSSP database of protein structure- sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. Fig. 6. Coloring scheme used to color residues by their relative importance. http://swift.cmbi.kun.nl/swift/hssp/

4.3.5 LaTex The text for this report was processed using LATEX; • rho ET score - the smaller this value, the lesser variability of Leslie Lamport, “LaTeX: A Document Preparation System Addison- this position across the branches of the tree (and, presumably, Wesley,” Reading, Mass. (1986). the greater the importance for the protein) 4.3.6 Muscle When making alignments “from scratch”, report • cvg coverage - percentage of the residues on the structure which maker uses Muscle alignment program: Edgar, Robert C. (2004), have this rho or smaller ”MUSCLE: multiple sequence alignment with high accuracy and • gaps percentage of gaps in this column high throughput.” Nucleic Acids Research 32(5), 1792-97. 4.2 Color schemes used http://www.drive5.com/muscle/ The following color scheme is used in figures with residues colored 4.3.7 Pymol The figures in this report were produced using by cluster size: black is a single-residue cluster; clusters composed of Pymol. The scripts can be found in the attachment. Pymol more than one residue colored according to this hierarchy (ordered is an open-source application copyrighted by DeLano Scien- by descending size): red, blue, yellow, green, purple, azure, tur- tific LLC (2005). For more information about Pymol see quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, http://pymol.sourceforge.net/. (Note for Windows bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, users: the attached package needs to be unzipped for Pymol to read DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, the scripts and launch the viewer.) tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated 4.4 Note about ET Viewer evolutionary pressure they experience can be seen in Fig. 6. Dan Morgan from the Lichtarge lab has developed a visualization 4.3 Credits 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 stati- http://mammoth.bcm.tmc.edu/traceview/ stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the The viewer is self-unpacking and self-installing. Input files to be used alignment length (e.g. including gap characters). Also shown are with ETV (extension .etvx) can be found in the attachment to the some percent identities. A percent pairwise alignment identity is defi- main report. ned as (idents / MIN(len1, len2)) where idents is the number of 4.5 Citing this work exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and The method used to rank residues and make predictions in this report ”most unrelated pair” of the alignment are the average, maximum, can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant Evolution-Entropy Hybrid Methods for Ranking of Protein Residues seq” is calculated by finding the maximum pairwise identity (best by Importance” J. Mol. Bio. 336: 1265-82. For the original version relative) for all N sequences, then finding the minimum of these N of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- numbers (hence, the most outlying sequence). alistat is copyrighted tionary Trace Method Defines Binding Surfaces Common to Protein by HHMI/Washington University School of Medicine, 1992-2001, Families” J. Mol. Bio. 257: 342-358. and freely distributed under the GNU General Public License. report maker itself is described in Mihalek I., I. Res and O. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 4.3.2 CE To map ligand binding sites from different of service for comparative analysis of proteins.” Bioinformatics source structures, report maker uses the CE program: 22:1656-7.

6 4.6 About report maker • 1akoA.cluster report.summary - Cluster report summary for report maker was written in 2006 by Ivana Mihalek. The 1D ran- 1akoA king visualization program was written by Ivica Res.ˇ report maker • 1akoA.ranks - Ranks file in sequence order for 1akoA is copyrighted by Lichtarge Lab, Baylor College of Medicine, • 1akoA.clusters - Cluster descriptions for 1akoA Houston. • 1akoA.msf - the multiple sequence alignment used for the chain 4.7 Attachments 1akoA The following files should accompany this report: • 1akoA.descr - description of sequences used in 1akoA msf • 1akoA.ranks sorted - full listing of residues and their ranking • 1akoA.complex.pdb - coordinates of 1akoA with all of its for 1akoA interacting partners • 1akoA.etvx - ET viewer input file for 1akoA

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