Pages 1–7 2vtv Evolutionary trace report by report maker December 4, 2009

4.3.1 Alistat 6 4.3.2 CE 6 4.3.3 DSSP 6 4.3.4 HSSP 6 4.3.5 LaTex 6 4.3.6 Muscle 6 4.3.7 Pymol 6 4.4 Note about ET Viewer 6 4.5 Citing this work 6 4.6 About report maker 6 4.7 Attachments 7

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2vtv): Title: Phaz7 depolymerase from paucimonas lemoignei CONTENTS Compound: Mol id: 1; molecule: phb depolymerase phaz7; chain: a, b; fragment: residues 39-380; ec: 3.1.1.75 1 Introduction 1 Organism, scientific name: Paucimonas Lemoignei 2vtv contains a single unique chain 2vtvA (340 residues long) and 2 Chain 2vtvA 1 its homologue 2vtvB. 2.1 Q939Q9 overview 1 2.2 Multiple sequence alignment for 2vtvA 1 2.3 Residue ranking in 2vtvA 1 2.4 Top ranking residues in 2vtvA and their position on the structure 2 2.4.1 Clustering of residues at 25% coverage. 2 2 CHAIN 2VTVA 2.4.2 Overlap with known functional surfaces at 2.1 Q939Q9 overview 25% coverage. 2 From SwissProt, id Q939Q9, 94% identical to 2vtvA: 2.4.3 Possible novel functional surfaces at 25% Description: PHB depolymerase PhaZ7 (Fragment). coverage. 3 Organism, scientific name: lemoignei. : ; ; ; Burkhol- 3 Notes on using trace results 5 deriales; ; Paucimonas. 3.1 Coverage 5 3.2 Known substitutions 5 3.3 Surface 5 3.4 Number of contacts 5 3.5 Annotation 5 2.2 Multiple sequence alignment for 2vtvA 3.6 Mutation suggestions 5 For the chain 2vtvA, the alignment 2vtvA.msf (attached) with 11 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 5 database, and fragments shorter than 75% of the query as well as 4.1 File formats 5 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 6 to this report, under the name of 2vtvA.msf. Its statistics, from the 4.3 Credits 6 alistat program are the following:

1 Lichtarge lab 2006 residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment.

Fig. 1. Residues 1-170 in 2vtvA colored by their relative importance. (See Appendix, Fig.9, for the coloring scheme.)

Fig. 2. Residues 171-342 in 2vtvA colored by their relative importance. (See Appendix, Fig.9, for the coloring scheme.) Fig. 3. Residues in 2vtvA, colored by their relative importance. Clockwise: front, back, top and bottom views.

Format: MSF Number of sequences: 11 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Total number of residues: 2793 top 25% of all residues, this time colored according to clusters they Smallest: 179 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 340 in Table 1. Average length: 253.9 Table 1. Alignment length: 340 cluster size member Average identity: 37% color residues Most related pair: 97% red 83 29,30,38,40,42,43,44,45,46 Most unrelated pair: 23% 47,48,49,50,51,53,55,74,77 Most distant seq: 30% 81,82,85,86,87,88,89,91,92 93,94,104,105,106,107,114 117,118,119,121,122,123,124 Furthermore, 4% of residues show as conserved in this alignment. 125,126,129,130,132,133,135 The alignment consists of sequences. (Descriptions of some 136,137,138,139,140,141,142 sequences were not readily available.) The file containing the 143,145,146,155,156,158,159 sequence descriptions can be found in the attachment, under the name 160,161,162,163,164,165,166 2vtvA.descr. 167,168,170,171,174,175,182 2.3 Residue ranking in 2vtvA 183,184,185,196,197,199,201 The 2vtvA sequence is shown in Figs. 1–2, with each residue colored Table 1. Clusters of top ranking residues in 2vtvA. according to its estimated importance. The full listing of residues in 2vtvA can be found in the file called 2vtvA.ranks sorted in the attachment. 2.4.2 Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier 2.4 Top ranking residues in 2vtvA and their position on and the heteroatom name used in that file. the structure Glycerol binding site. Table 2 lists the top 25% of residues at the In the following we consider residues ranking among top 25% of resi- interface with 2vtvAGOL1343 (glycerol). The following table (Table dues in the protein . Figure 3 shows residues in 2vtvA colored by their 3) suggests possible disruptive replacements for these residues (see importance: bright red and yellow indicate more conserved/important Section 3.6).

2 Table 2. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 166 R L(45) 0.18 14/0 2.67 site Y(27) R(27)

Table 2. The top 25% of residues in 2vtvA at the interface with glyce- rol.(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 num- ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 3. res type disruptive mutations 166 R (TD)(E)(VCAG)(S)

Table 3. List of disruptive mutations for the top 25% of residues in 2vtvA, that are at the interface with glycerol. Fig. 4. Residues in 2vtvA, 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.

Fig. 5. Residues in 2vtvA, at the interface with glycerol, colored by their relative importance. The ligand (glycerol) is colored green. Atoms further than 30A˚ 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 2vtvA.)

Figure 5 shows residues in 2vtvA colored by their importance, at the interface with 2vtvAGOL1343. Interface with 2vtvB.Table 4 lists the top 25% of residues at the interface with 2vtvB. The following table (Table 5) suggests possible disruptive replacements for these residues (see Section 3.6).

3 Table 4. shows (in blue) the rest of the larger cluster this surface belongs to. res type subst’s cvg noc/ dist (%) bb (A˚ ) 201 G S(36) 0.24 4/4 4.16 .(9) Y(27) G(27)

Table 4. The top 25% of residues in 2vtvA at the interface with 2vtvB. (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. 7. A possible active surface on the chain 2vtvA. The larger cluster it belongs to is shown in blue. Table 5. res type disruptive mutations The residues belonging to this surface ”patch” are listed in Table 201 G (K)(R)(E)(QM) 6, while Table 7 suggests possible disruptive replacements for these residues (see Section 3.6). Table 5. List of disruptive mutations for the top 25% of residues in 2vtvA, Table 6. that are at the interface with 2vtvB. res type substitutions(%) cvg antn 49 N N(100) 0.04 136 S S(100) 0.04 163 G G(100) 0.04 182 P P(90)S(9) 0.09 199 P P(90).(9) 0.09 146 L I(72)L(27) 0.12 168 L L(90)I(9) 0.12 50 G S(63)T(9)G(27) 0.13 142 S A(90)S(9) 0.13 164 G A(90)G(9) 0.13 104 N Q(36)N(63) 0.14 107 S S(81)D(18) 0.15 137 M M(81)L(18) 0.15 162 A A(90)G(9) 0.15 185 G G(63)S(36) 0.16 105 Y Y(63)N(9)S(27) 0.17 175 G G(63)P(9)T(27) 0.17 166 R L(45)Y(27)R(27) 0.18 site 197 F L(36).(9)F(54) 0.23 174 T A(36)Y(9)S(27) 0.24 T(27) 201 G S(36).(9)Y(27) 0.24 G(27) 102 Q Q(54)A(9)S(27) 0.25 Fig. 6. Residues in 2vtvA, at the interface with 2vtvB, colored by their rela- N(9) tive importance. 2vtvB is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 2vtvA.) Table 6. Residues forming surface ”patch” in 2vtvA.

Figure 6 shows residues in 2vtvA colored by their importance, at the Table 7. interface with 2vtvB. res type disruptive mutations 2.4.3 Possible novel functional surfaces at 25% coverage. One 49 N (Y)(FTWH)(SEVCARG)(MD) group of residues is conserved on the 2vtvA surface, away from (or continued in next column susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 2vtv. It is shown in Fig. 7. The right panel

4 Table 7. continued Table 8. continued res type disruptive res type substitutions(%) cvg mutations 119 D E(72)Q(9)D(18) 0.19 136 S (KR)(FQMWH)(NYELPI)(D) 122 K K(45)L(54) 0.19 163 G (KER)(FQMWHD)(NYLPI)(SVA) 38 A V(27)A(63)F(9) 0.20 182 P (R)(Y)(H)(K) 40 K N(9)K(81)Q(9) 0.20 199 P (YR)(TH)(SCG)(KE) 156 R D(63)A(27)R(9) 0.21 146 L (YR)(TH)(SKECG)(FQWD) 123 A A(81)S(9)I(9) 0.22 168 L (YR)(TH)(SKECG)(FQWD) 85 C S(27)A(27)C(45) 0.23 50 G (KR)(E)(FQMWH)(D) 126 G G(54)C(9)K(27) 0.24 142 S (KR)(QH)(FYEMW)(N) N(9) 164 G (KER)(QHD)(FYMW)(N) 104 N (Y)(FTWH)(SVCAG)(ER) Table 8. Residues forming surface ”patch” in 2vtvA. 107 S (R)(K)(FWH)(QM) 137 M (Y)(TH)(R)(SCG) 162 A (KER)(Y)(QHD)(N) Table 9. 185 G (KR)(E)(FQMWH)(D) res type disruptive 105 Y (K)(M)(QR)(E) mutations 175 G (R)(K)(E)(H) 81 G (KER)(FQMWHD)(NYLPI)(SVA) 166 R (TD)(E)(VCAG)(S) 82 Y (K)(QM)(NEVLAPIR)(D) 197 F (KE)(T)(QD)(CG) 124 Y (K)(QM)(R)(NELPI) 174 T (K)(R)(Q)(M) 125 T (KR)(FQMWH)(NELPI)(D) 201 G (K)(R)(E)(QM) 129 Q (Y)(FTW)(H)(SVCAG) 102 Q (Y)(H)(FW)(T) 119 D (FWHR)(Y)(VCAG)(T) 122 K (Y)(T)(FW)(SCG) Table 7. Disruptive mutations for the surface patch in 2vtvA. 38 A (KE)(R)(Y)(D) 40 K (Y)(FTW)(S)(VCAG) 156 R (TY)(D)(ECG)(FVLAWPI) Another group of surface residues is shown in Fig.8. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. 123 A (R)(KY)(E)(H) 85 C (KR)(E)(QH)(FMW) 126 G (E)(FWR)(H)(KYD)

Table 9. Disruptive mutations for the surface patch in 2vtvA.

3 NOTES ON USING TRACE RESULTS 3.1 Coverage Trace results are commonly expressed in terms of coverage: the resi- due 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 Fig. 8. Another possible active surface on the chain 2vtvA. The larger cluster it belongs to is shown in blue. 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 The residues belonging to this surface ”patch” are listed in Table couple of residues should affect the protein somehow, with the exact 8, while Table 9 suggests possible disruptive replacements for these effects to be determined experimentally. residues (see Section 3.6). 3.2 Known substitutions Table 8. One of the table columns is “substitutions” - other amino acid types res type substitutions(%) cvg seen at the same position in the alignment. These amino acid types 81 G G(100) 0.04 may be interchangeable at that position in the protein, so if one wants 82 Y Y(100) 0.04 to affect the protein by a point mutation, they should be avoided. For 124 Y Y(90)S(9) 0.09 example if the substitutions are “RVK” and the original protein has 125 T T(90)S(9) 0.09 an R at that position, it is advisable to try anything, but RVK. Conver- 129 Q K(81)Q(18) 0.18 sely, when looking for substitutions which will not affect the protein, continued in next column one may try replacing, R with K, or (perhaps more surprisingly), with V. The percentage of times the substitution appears in the alignment

5 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 Surface COVERAGE

To detect candidates for novel functional interfaces, first we look for V residues that are solvent accessible (according to DSSP program) by 2 100% 50% 30% 5% at least 10A˚ , which is roughly the area needed for one water mole- cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor within 5A˚ 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 V equally important in maintaining the interaction specificity - they RELATIVE IMPORTANCE should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) Fig. 9. Coloring scheme used to color residues by their relative importance. 3.4 Number of contacts Another column worth noting is denoted “noc/bb”; it tells the num- • alignment# number of the position in the alignment ber of contacts heavy atoms of the residue in question make across • residue# residue number in the PDB file the interface, as well as how many of them are realized through the • type amino acid type backbone atoms (if all or most contacts are through the backbone, • mutation presumably won’t have strong impact). Two heavy atoms rank rank of the position according to older version of ET are considered to be “in contact” if their centers are closer than 5A˚ . • variability has two subfields: 1. number of different amino acids appearing in in this column 3.5 Annotation of the alignment If the residue annotation is available (either from the pdb file or 2. their type from other sources), another column, with the header “annotation” • rho ET score - the smaller this value, the lesser variability of appears. Annotations carried over from PDB are the following: site this position across the branches of the tree (and, presumably, (indicating existence of related site record in PDB ), S-S (disulfide the greater the importance for the protein) bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue). • cvg coverage - percentage of the residues on the structure which have this rho or smaller 3.6 Mutation suggestions • gaps percentage of gaps in this column Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that 4.2 Color schemes used they are meant to be disruptive to the interaction of the protein The following color scheme is used in figures with residues colored with its ligand. The attempt is made to complement the following by cluster size: black is a single-residue cluster; clusters composed of properties: small [AV GSTC], medium [LPNQDEMIK], large more than one residue colored according to this hierarchy (ordered [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- by descending size): red, blue, yellow, green, purple, azure, tur- tively [KHR], or negatively [DE] charged, aromatic [WFYH], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, long aliphatic chain [EKRQM], OH-group possession [SDETY ], bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, and NH2 group possession [NQRK]. The suggestions are listed DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, according to how different they appear to be from the original amino tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. acid, and they are grouped in round brackets if they appear equally The colors used to distinguish the residues by the estimated disruptive. From left to right, each bracketed group of amino acid evolutionary pressure they experience can be seen in Fig. 9. types resembles more strongly the original (i.e. is, presumably, less disruptive) These suggestions are tentative - they might prove disrup- 4.3 Credits tive to the fold rather than to the interaction. Many researcher will 4.3.1 Alistat alistat reads a multiple sequence alignment from the choose, however, the straightforward alanine mutations, especially in file and shows a number of simple statistics about it. These stati- the beginning stages of their investigation. stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the 4 APPENDIX alignment length (e.g. including gap characters). Also shown are some percent identities. A percent pairwise alignment identity is defi- 4.1 File formats ned as (idents / MIN(len1, len2)) where idents is the number of Files with extension “ranks sorted” are the actual trace results. The exact identities and len1, len2 are the unaligned lengths of the two fields in the table in this file: sequences. The ”average percent identity”, ”most related pair”, and

6 ”most unrelated pair” of the alignment are the average, maximum, http://mammoth.bcm.tmc.edu/traceview/ and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant seq” is calculated by finding the maximum pairwise identity (best The viewer is self-unpacking and self-installing. Input files to be used relative) for all N sequences, then finding the minimum of these N with ETV (extension .etvx) can be found in the attachment to the numbers (hence, the most outlying sequence). alistat is copyrighted main report. by HHMI/Washington University School of Medicine, 1992-2001, and freely distributed under the GNU General Public License. 4.5 Citing this work 4.3.2 CE To map ligand binding sites from different The method used to rank residues and make predictions in this report source structures, report maker uses the CE program: can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) Evolution-Entropy Hybrid Methods for Ranking of Protein Residues ”Protein structure alignment by incremental combinatorial extension by Importance” J. Mol. Bio. 336: 1265-82. For the original version (CE) of the optimal path . Protein Engineering 11(9) 739-747. of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- 4.3.3 DSSP In this work a residue is considered solvent accessi- tionary Trace Method Defines Binding Surfaces Common to Protein ble if the DSSP program finds it exposed to water by at least 10A˚ 2, Families” J. Mol. Bio. 257: 342-358. which is roughly the area needed for one water molecule to come in report maker itself is described in Mihalek I., I. Res and O. the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version of service for comparative analysis of proteins.” Bioinformatics by [email protected] November 18,2002, 22:1656-7. http://www.cmbi.kun.nl/gv/dssp/descrip.html. 4.6 About report maker 4.3.4 HSSP Whenever available, report maker uses HSSP ali- report maker was written in 2006 by Ivana Mihalek. The 1D ran- gnment as a starting point for the analysis (sequences shorter than king visualization program was written by Ivica Res.ˇ report maker 75% of the query are taken out, however); R. Schneider, A. de is copyrighted by Lichtarge Lab, Baylor College of Medicine, Daruvar, and C. Sander. ”The HSSP database of protein structure- Houston. sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. http://swift.cmbi.kun.nl/swift/hssp/ 4.7 Attachments 4.3.5 LaTex The text for this report was processed using LATEX; The following files should accompany this report: Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). • 2vtvA.complex.pdb - coordinates of 2vtvA with all of its inter- 4.3.6 Muscle When making alignments “from scratch”, report acting partners maker uses Muscle alignment program: Edgar, Robert C. (2004), • 2vtvA.etvx - ET viewer input file for 2vtvA ”MUSCLE: multiple sequence alignment with high accuracy and • 2vtvA.cluster report.summary - Cluster report summary for high throughput.” Nucleic Acids Research 32(5), 1792-97. 2vtvA http://www.drive5.com/muscle/ • 2vtvA.ranks - Ranks file in sequence order for 2vtvA • 4.3.7 Pymol The figures in this report were produced using 2vtvA.clusters - Cluster descriptions for 2vtvA Pymol. The scripts can be found in the attachment. Pymol • 2vtvA.msf - the multiple sequence alignment used for the chain is an open-source application copyrighted by DeLano Scien- 2vtvA tific LLC (2005). For more information about Pymol see • 2vtvA.descr - description of sequences used in 2vtvA msf http://pymol.sourceforge.net/. (Note for Windows • 2vtvA.ranks sorted - full listing of residues and their ranking for users: the attached package needs to be unzipped for Pymol to read 2vtvA the scripts and launch the viewer.) • 2vtvA.2vtvAGOL1343.if.pml - Pymol script for Figure 5 4.4 Note about ET Viewer • 2vtvA.cbcvg - used by other 2vtvA – related pymol scripts Dan Morgan from the Lichtarge lab has developed a visualization • 2vtvA.2vtvB.if.pml - Pymol script for Figure 6 tool specifically for viewing trace results. If you are interested, please visit:

7