Transport Inference Parser: Inferring Transport Reactions from Protein Data for PGDBs

1. Transport Inference Parser:Inferring Transport Reactions from Protein Data for PGDBs Thomas J Lee, Peter Karp, AIC BRG Ian Paulsen consulting

2. Running the Transport Inference Parser 1. Run Pathway Tools. 2. Make the organism of interest the current organism. 3. [Run operon predictor]. 4. Select Tools/Pathologic. 5. From Pathologic, select Refine/Transport Inference Parser. 6. If running TIP for the first time on the organism, optionally provide its aerobicity. 7. Wait and observe progress. 8. When complete, Probable Transporter Table window appears. 9. You may now review and modify the inferred transporters.

3. Task Description Infer transport reactions from protein data and construct them in BioCyc KBs for a variety of organisms, automatically where possible, with human assistance where necessary.

4. Scope • Run for all Tier 3 KBs (~700 KB) • To support both automated and user-controlled operation: • Distinguish high- and low-confidence inferences • Automated mode accepts all high-confidence inferences • Track evidence where possible • Provide accept/reject/edit options to user

5. Output Construct the following for each inferred transported substrate: • Transport-Reaction frame of correct subclass • Assign compartments – use simple assumptions • Enzymatic-Reaction frame linking protein to reaction Construct Protein-Complexes as required

6. Sequence of operations 1. Find candidate transporter proteins. 2. Filter out candidates. 3. Identify substrate(s). 4. Assign an energy coupling to transporter. 5. Identify compartment of each substrate. 6. Group subunits of transporter complexes. 7. Construct full compartmental reaction from substrate and coupling. 8. Construct enzymatic reaction linking each reaction with protein.

7. 1. Find candidate transporter proteins • Input: all protein frames of organism • Output: internal data structure (PARTRANS) • Exclude proteins with long annotations (default: 12 words) • Tokenize the annotation • Annotation must contain an indicator. Exs:"transport”, “export”, “permease”, “channel”

8. 2. Filter candidates • Exclude if annotation matches a list of regular expressions of counterindicator phrases and patterns • Ex:“transport associated domain” • Exclude if annotation containscounterindicator word • Exs: “regulator”, “nuclear-export”

9. 3. Identify substrate(s) Search annotation for names of MetaCyc compounds. Details: Multiple substrates indicate multiple reactions, symport/antiport pair, or both. Exs: “cytosine/purines/uracil/thiamine/allantoin permease family protein” “magnesium and cobalt transport protein cora, putative” “sodium:sulfate symporter transmembrane domain protein” “probable agcs sodium/alanine/glycine symporter” Exclude non-substrates that look like compounds via an exception list. Exs: “as”“be”“c”“i”

10. 3. Identify substrate(s) (cont.) Name canonicalization. Ex: strip plurals. Affixed substrates. Exs: “-transporting”“-specific” Lookup special ionic forms. Exs: “cuprous”“ferric”“hydrogen” Resolve multivalent options using aerobicity. Exs: “FE”“CR”“MN” Two-word substrates, substrate classes. Ex: “amino acid”

11. 4. Assign an energy coupling. 1. Search annotation for prioritized list of indicators. Exs: ("atp-binding" . ATP) ("mfs" . SECONDARY) ("pts" . PTS) ("phosphotransferase" . PTS) ("carrier" . SECONDARY) ("channel" . CHANNEL) 2. Some substrates imply a coupling. Ex: protoheme => ATP Absence of indicator => UNKNOWN Deferred some more sophisticated techniques: • BLAST vs. E.coli • HMM family identification

12. 5. Identify compartment of each substrate. Use keywords to determine compartment of primary substrate (Exs: “export”, “antiporter”) Otherwise assume primary substrate is transported into cell (periplasm => cytoplasm) Deferred complex compartment analysis: • Assume E.coli-like cellular structure

13. 6. Group subunits of transporter complexes. Many transporters are systems of several proteins. These are grouped into complexes Grouping criteria; all must be met: • Predicted coupling is ATP or PEP • Predicted substrates are identical • Genes of proteins have a common operon (NOTE requirement on operon availability) Resulting complex is added to KB under Protein-Complexes.

14. 7. Construct full compartmental reaction from substrate and coupling. Determine set of transported substrates for this transporter: • For SECONDARY coupling: • Identify auxiliary substrate providing ion gradient (H+, Na+) • Remove from transported substrate list • Place on side of reaction indicated by symport/antiport clues • For other couplings: • Determined previously in substrate analysis

15. 7. Construct full compartmental reaction from substrate and coupling (cont). For each transported substrate of this transporter, either import reaction (from E.coli) or to create new one. • Search importKB for reaction with matching substrates (find-rxn-by-substrates) • Transported substrate added with indicated compartment • Auxiliary substrates determined by coupling. Ex: • CHANNEL typically have none • ATP have ATP/H2O  ADP/phosphate • If one reaction is found, import: (import-reactions trxns src-kb dst-kb …) • If multiple reactions found, retain all. • Else if reaction is not present in KB, create new rxn

16. 7. Construct full compartmental reaction from substrate and coupling (cont). Create new reaction: • Create reaction frame, subclass determined by coupling: • (create-instance-w-generated-id rxn-class) • Add transported and auxiliary substrates to appropriate sides of reaction

17. 8. Construct enzymatic reaction linking each reaction with protein. For each created reaction: • (add-reactions-to-protein …) • Added evidence code, history string arguments • Subordinates new [(import-reactions) handles import of enzymatic-reactions]

18. Running the Transport Inference Parser 1. Run Pathway Tools. 2. Make the organism of interest the current organism. 3. [Run operon predictor]. 4. Select Tools/Pathologic. 5. From Pathologic, select Refine/Transport Inference Parser. 6. If running TIP for the first time on the organism, optionally provide its aerobicity. 7. Wait and observe progress. 8. When complete, Probable Transporter Table window appears. 9. You may now review and modify the inferred transporters.

19. GUI Overview • Window is titled: Probable Transporter Table for Organism • Table of inferred transporters is organized into columns: • Status • Gene • Substrate • Coupling • Reaction / Function 3. Each row contains a transport reaction description: • Multiple reactions per transport protein are possible • Sort by Gene (the default) to keep together visually 4. Aggregate pane shows counts by status. 5. Mousing over a reaction shows details in bottom pane.

20. Notional GUI Example

21. Reviewing and Editing • Left-click on a row • Dialog box appears • May edit: • Function (name) • Energy coupling • May invoke Reaction Editor on reaction • May retract reaction • May update status

22. Transporter Status • Accepted: • Incorporate transporter into PGDB upon save • Rejected: • Discard transporter upon save • Unreviewed: • Initial value of status • Change to Accepted to preserve edits Accept and Reject are undoable

23. Filtering and Sorting • Filtering excluded transporters from display: • Filter low- or high-confidence transporters • Filter by status • Filter by number of reactions per substrate • Sort transporters by: • Gene • Energy Coupling • Substrate number/name • Status (e.g., Accepted, Rejected)

24. Group Operations TIP permits en masse acceptance or rejection of remaining predictions being shown: Edit / Accept all Unreviewed predictions being shown Edit / Accept all Unreviewed predictions being shown

25. Saving Your Work The TIP has made in-memory modifications to the KB; nothing has been saved. Exit / Save saves all predictions & edits. Exit / Cancel reverts to most recent save.

26. Multisession Workflow • TIP remembers accepted predictions in the KB. • TIP remembers rejected transporters in a file under the organism directory. • To continue, re-run TIP and resume session. • If you don’t resume (i.e., start from scratch): • Will not re-predict Accepteds • Will re-predict Rejecteds