Pages 1–6 1lbe Evolutionary trace report by report maker November 14, 2009

4.3.1 Alistat 5 4.3.2 CE 5 4.3.3 DSSP 5 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 6

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1lbe): Title: adp ribosyl cyclase Compound: Mol id: 1; molecule: adp ribosyl cyclase; chain: a, b; synonym: cyclase, nadase; ec: 3.2.2.5 Organism, scientific name: Aplysia Californica; 1lbe contains a single unique chain 1lbeA (250 residues long) and its homologue 1lbeB.

CONTENTS 2 CHAIN 1LBEA 2.1 P29241 overview 1 Introduction 1 From SwissProt, id P29241, 100% identical to 1lbeA: 2 Chain 1lbeA 1 Description: ADP-ribosyl cyclase precursor (EC 3.2.2.5) (NAD(+) 2.1 P29241 overview 1 nucleosidase) (NADase) (NAD glycohydrolase) (ADRC). 2.2 Multiple sequence alignment for 1lbeA 1 Organism, scientific name: Aplysia californica (California sea 2.3 Residue ranking in 1lbeA 1 hare). 2.4 Top ranking residues in 1lbeA and their position on Taxonomy: Eukaryota; Metazoa; ; ; Ortho- the structure 1 gastropoda; ; ; Euthyneura; Opistho- 2.4.1 Clustering of residues at 25% coverage. 2 branchia; ; Aplysioidea; ; Aplysia. 2.4.2 Overlap with known functional surfaces at Function: Synthesizes cyclic ADP-ribose, a second messenger for 25% coverage. 3 calcium mobilization from endoplasmic reticulum. 2.4.3 Possible novel functional surfaces at 25% Catalytic activity: NAD(+) + H(2)O = ADP-ribose + nicotinamide. coverage. 3 Enzyme regulation: Activity is presumably regulated by its seque- stration in vesicles before egg fertilization. After fertilization and 3 Notes on using trace results 4 upon NADase release, it could then be regulated via its potential 3.1 Coverage 4 phosphorylation sites. 3.2 Known substitutions 4 Subcellular location: Localized to vesicles or granules within ova of 3.3 Surface 4 all stages. 3.4 Number of contacts 5 Tissue specificity: Oocytes. 3.5 Annotation 5 Developmental stage: Immature eggs have higher levels of NADase 3.6 Mutation suggestions 5 transcripts than the mature ones. Ptm: Has different isoforms which may be the result of different 4 Appendix 5 amounts of phosphorylation. 4.1 File formats 5 Similarity: Belongs to the ADP-ribosyl cyclase family. 4.2 Color schemes used 5 About: This Swiss-Prot entry is copyright. It is produced through a 4.3 Credits 5 collaboration between the Swiss Institute of Bioinformatics and the

1 Lichtarge lab 2006 2.4 Top ranking residues in 1lbeA and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 1lbeA 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. 1. Residues 1-125 in 1lbeA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Fig. 2. Residues 126-250 in 1lbeA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

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 Multiple sequence alignment for 1lbeA For the chain 1lbeA, the alignment 1lbeA.msf (attached) with 14 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 Fig. 3. Residues in 1lbeA, colored by their relative importance. Clockwise: to this report, under the name of 1lbeA.msf. Its statistics, from the front, back, top and bottom views. alistat program are the following:

Format: MSF 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Number of sequences: 14 top 25% of all residues, this time colored according to clusters they Total number of residues: 3389 belong to. The clusters in Fig.4 are composed of the residues listed Smallest: 230 in Table 1. Largest: 250 Table 1. Average length: 242.1 cluster size member Alignment length: 250 color residues Average identity: 44% red 62 12,14,15,18,34,41,48,50,51 Most related pair: 95% 54,57,58,61,70,71,73,76,77 Most unrelated pair: 30% 83,84,97,99,101,102,103,107 Most distant seq: 45% 109,111,112,113,125,126,131 140,144,147,148,151,152,155 Furthermore, 10% of residues show as conserved in this alignment. 158,159,160,161,162,169,173 The alignment consists of 92% eukaryotic ( 85% vertebrata) 175,176,179,183,193,194,196 sequences. (Descriptions of some sequences were not readily availa- 206,214,215,227,231,237,239 ble.) The file containing the sequence descriptions can be found in 248 the attachment, under the name 1lbeA.descr. Table 1. Clusters of top ranking residues in 1lbeA. 2.3 Residue ranking in 1lbeA The 1lbeA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues 2.4.2 Overlap with known functional surfaces at 25% coverage. in 1lbeA can be found in the file called 1lbeA.ranks sorted in the The name of the ligand is composed of the source PDB identifier attachment. and the heteroatom name used in that file.

2 Table 3. continued res type disruptive mutations 239 C (E)(D)(FKMW)(YQLPHIR) 248 C (KER)(FQMWHD)(NLPI)(Y) 237 L (YR)(H)(TKE)(SQCDG)

Table 3. List of disruptive mutations for the top 25% of residues in 1lbeA, that are at the interface with 1lbeB.

Fig. 4. Residues in 1lbeA, 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.

Interface with 1lbeB.Table 2 lists the top 25% of residues at the interface with 1lbeB. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6). Table 2. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) Fig. 5. Residues in 1lbeA, at the interface with 1lbeB, colored by their rela- 14 R R(100) 0.10 47/4 2.88 tive importance. 1lbeB is shown in backbone representation (See Appendix 109 L L(100) 0.10 3/0 3.71 for the coloring scheme for the protein chain 1lbeA.) 239 C R(7) 0.14 25/12 3.57 S-S C(92) 248 C .(7) 0.14 17/13 2.42 S-S Figure 5 shows residues in 1lbeA colored by their importance, at the C(92) interface with 1lbeB. 237 L L(92) 0.20 11/6 3.82 V(7) 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1lbeA surface, away from (or susbtantially larger than) other functional sites and interfaces reco- Table 2. The top 25% of residues in 1lbeA at the interface with 1lbeB. gnizable in PDB entry 1lbe. It is shown in Fig. 6. The right panel (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 shows (in blue) the rest of the larger cluster this surface belongs to. in the bracket; noc/bb: number of contacts with the ligand, with the number of The residues belonging to this surface ”patch” are listed in Table contacts realized through backbone atoms given in the bracket; dist: distance 4, while Table 5 suggests possible disruptive replacements for these of closest apporach to the ligand. ) residues (see Section 3.6). Table 4. Table 3. res type substitutions(%) cvg antn res type disruptive 14 R R(100) 0.10 mutations 18 Y Y(100) 0.10 14 R (TD)(SYEVCLAPIG)(FMW)(N) 34 C C(100) 0.10 S-S 109 L (YR)(TH)(SKECG)(FQWD) 48 K K(100) 0.10 50 P P(100) 0.10 continued in next column continued in next column

3 Table 5. continued res type disruptive mutations 77 W (KE)(TQD)(SNCRG)(M) 97 L (YR)(TH)(SKECG)(FQWD) 99 D (R)(FWH)(KYVCAG)(TQM) 109 L (YR)(TH)(SKECG)(FQWD) 112 C (KER)(FQMWHD)(NYLPI)(SVA) 125 C (KER)(FQMWHD)(NYLPI)(SVA) 126 P (YR)(TH)(SKECG)(FQWD) 131 C (KER)(FQMWHD)(NYLPI)(SVA) 140 W (KE)(TQD)(SNCRG)(M) Fig. 6. A possible active surface on the chain 1lbeA. The larger cluster it 144 S (KR)(FQMWH)(NYELPI)(D) belongs to is shown in blue. 179 E (FWH)(YVCARG)(T)(SNKLPI) 58 Y (K)(Q)(EM)(NR) Table 4. continued 101 L (R)(TY)(KE)(SCHG) res type substitutions(%) cvg antn 111 W (KE)(TQD)(SNCRG)(M) 51 C C(100) 0.10 S-S 175 F (KE)(T)(QDR)(SCG) 77 W W(100) 0.10 183 L (Y)(R)(TH)(SCG) 97 L L(100) 0.10 12 L (Y)(THR)(SCG)(FEW) 99 D D(100) 0.10 147 Y (K)(Q)(EM)(NR) 109 L L(100) 0.10 57 S (R)(K)(FWH)(QM) 112 C C(100) 0.10 S-S 107 N (Y)(FWH)(TR)(VCAG) 125 C C(100) 0.10 S-S 173 S (KR)(FQMWH)(E)(NYLPI) 126 P P(100) 0.10 169 Y (K)(EQM)(NVLAPDI)(R) 131 C C(100) 0.10 S-S 54 D (R)(H)(FW)(K) 140 W W(100) 0.10 76 F (KE)(T)(QDR)(SCG) 144 S S(100) 0.10 176 G (E)(D)(K)(R) 179 E E(100) 0.10 58 Y Y(92)F(7) 0.13 Table 5. Disruptive mutations for the surface patch in 1lbeA. 101 L L(92)F(7) 0.13 111 W W(92)F(7) 0.13 175 F F(92)L(7) 0.13 183 L L(92)M(7) 0.13 3 NOTES ON USING TRACE RESULTS 12 L Q(7)L(92) 0.14 3.1 Coverage 147 Y F(57)Y(42) 0.15 57 S D(85)S(14) 0.18 Trace results are commonly expressed in terms of coverage: the resi- 107 N D(85)N(14) 0.18 due is important if its “coverage” is small - that is if it belongs to 173 S S(71)G(28) 0.19 some small top percentage of residues [100% is all of the residues 169 Y F(50)Y(42)R(7) 0.20 in a chain], according to trace. The ET results are presented in the 54 D T(57)L(28)D(14) 0.22 form of a table, usually limited to top 25% percent of residues (or 76 F F(78)L(21) 0.24 to some nearby percentage), sorted by the strength of the presumed 176 G G(64)A(28)R(7) 0.25 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 Table 4. Residues forming surface ”patch” in 1lbeA. effects to be determined experimentally.

3.2 Known substitutions Table 5. res type disruptive One of the table columns is “substitutions” - other amino acid types mutations seen at the same position in the alignment. These amino acid types 14 R (TD)(SYEVCLAPIG)(FMW)(N) may be interchangeable at that position in the protein, so if one wants 18 Y (K)(QM)(NEVLAPIR)(D) to affect the protein by a point mutation, they should be avoided. For 34 C (KER)(FQMWHD)(NYLPI)(SVA) example if the substitutions are “RVK” and the original protein has 48 K (Y)(FTW)(SVCAG)(HD) an R at that position, it is advisable to try anything, but RVK. Conver- 50 P (YR)(TH)(SKECG)(FQWD) sely, when looking for substitutions which will not affect the protein, 51 C (KER)(FQMWHD)(NYLPI)(SVA) one may try replacing, R with K, or (perhaps more surprisingly), with continued in next column 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

4 guide - due to rounding errors these percentages often do not add up to 100%. 3.3 Surface To detect candidates for novel functional interfaces, first we look for COVERAGE residues that are solvent accessible (according to DSSP program) by ˚ 2 at least 10A , which is roughly the area needed for one water mole- V cule to come in the contact with the residue. Furthermore, we require 100% 50% 30% 5% 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 equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- V sing the set for mutagenesis. (Especially if they form a cluster with RELATIVE IMPORTANCE the surface residues.) 3.4 Number of contacts Fig. 7. Coloring scheme used to color residues by their relative importance. Another column worth noting is denoted “noc/bb”; it tells the num- ber of contacts heavy atoms of the residue in question make across 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 appears. Annotations carried over from PDB are the following: site ET score - the smaller this value, the lesser variability of (indicating existence of related site record in PDB ), S-S (disulfide this position across the branches of the tree (and, presumably, bond forming residue), hb (hydrogen bond forming residue, jb (james the greater the importance for the protein) 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. 7. 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 choose, however, the straightforward alanine mutations, especially in 4.3.1 Alistat alistat reads a multiple sequence alignment from the the beginning stages of their investigation. file and shows a number of simple statistics about it. These stati- 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 4.1 File formats some percent identities. A percent pairwise alignment identity is defi- Files with extension “ranks sorted” are the actual trace results. The ned as (idents / MIN(len1, len2)) where idents is the number of fields in the table in this file: exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and • alignment# number of the position in the alignment ”most unrelated pair” of the alignment are the average, maximum, • residue# residue number in the PDB file and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant

5 seq” is calculated by finding the maximum pairwise identity (best http://mammoth.bcm.tmc.edu/traceview/ relative) for all N sequences, then finding the minimum of these N numbers (hence, the most outlying sequence). alistat is copyrighted The viewer is self-unpacking and self-installing. Input files to be used by HHMI/Washington University School of Medicine, 1992-2001, with ETV (extension .etvx) can be found in the attachment to the and freely distributed under the GNU General Public License. main report. 4.3.2 CE To map ligand binding sites from different source structures, report maker uses the CE program: 4.5 Citing this work http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) The method used to rank residues and make predictions in this report ”Protein structure alignment by incremental combinatorial extension can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of (CE) of the optimal path . Protein Engineering 11(9) 739-747. Evolution-Entropy Hybrid Methods for Ranking of Protein Residues 4.3.3 DSSP In this work a residue is considered solvent accessi- by Importance” J. Mol. Bio. 336: 1265-82. For the original version ble if the DSSP program finds it exposed to water by at least 10A˚ 2, of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- which is roughly the area needed for one water molecule to come in tionary Trace Method Defines Binding Surfaces Common to Protein the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Families” J. Mol. Bio. 257: 342-358. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version report maker itself is described in Mihalek I., I. Res and O. by [email protected] November 18,2002, Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type of service for comparative analysis of proteins.” Bioinformatics http://www.cmbi.kun.nl/gv/dssp/descrip.html. 22:1656-7. 4.3.4 HSSP Whenever available, report maker uses HSSP ali- gnment as a starting point for the analysis (sequences shorter than 4.6 About report maker 75% of the query are taken out, however); R. Schneider, A. de report maker was written in 2006 by Ivana Mihalek. The 1D ran- Daruvar, and C. Sander. ”The HSSP database of protein structure- king visualization program was written by Ivica Res.ˇ report maker sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. is copyrighted by Lichtarge Lab, Baylor College of Medicine, Houston. http://swift.cmbi.kun.nl/swift/hssp/

4.3.5 LaTex The text for this report was processed using LATEX; 4.7 Attachments Leslie Lamport, “LaTeX: A Document Preparation System Addison- The following files should accompany this report: Wesley,” Reading, Mass. (1986).

4.3.6 Muscle When making alignments “from scratch”, report • 1lbeA.complex.pdb - coordinates of 1lbeA with all of its inter- maker uses Muscle alignment program: Edgar, Robert C. (2004), acting partners ”MUSCLE: multiple sequence alignment with high accuracy and • 1lbeA.etvx - ET viewer input file for 1lbeA high throughput.” Nucleic Acids Research 32(5), 1792-97. • 1lbeA.cluster report.summary - Cluster report summary for http://www.drive5.com/muscle/ 1lbeA 4.3.7 Pymol The figures in this report were produced using • 1lbeA.ranks - Ranks file in sequence order for 1lbeA Pymol. The scripts can be found in the attachment. Pymol • 1lbeA.clusters - Cluster descriptions for 1lbeA is an open-source application copyrighted by DeLano Scien- • 1lbeA.msf - the multiple sequence alignment used for the chain tific LLC (2005). For more information about Pymol see 1lbeA http://pymol.sourceforge.net/. (Note for Windows • users: the attached package needs to be unzipped for Pymol to read 1lbeA.descr - description of sequences used in 1lbeA msf the scripts and launch the viewer.) • 1lbeA.ranks sorted - full listing of residues and their ranking for 1lbeA 4.4 Note about ET Viewer • 1lbeA.1lbeB.if.pml - Pymol script for Figure 5 Dan Morgan from the Lichtarge lab has developed a visualization • tool specifically for viewing trace results. If you are interested, please 1lbeA.cbcvg - used by other 1lbeA – related pymol scripts visit:

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