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FROM SIMPLE PEPTIDES TO MULTI-COMPONENT METABOLONS

FROM SIMPLE PEPTIDES TO MULTI-COMPONENT METABOLONS. Milton Saier Division of Biological Sciences University of California, San Diego msaier@ucsd.edu. Outline. Introduction: The Power of Bioinformatics From Peptides to Carriers: Mapping Evolutionary Pathways

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FROM SIMPLE PEPTIDES TO MULTI-COMPONENT METABOLONS

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  1. FROM SIMPLE PEPTIDES TO MULTI-COMPONENT METABOLONS Milton Saier Division of Biological Sciences University of California, San Diego msaier@ucsd.edu

  2. Outline • Introduction: The Power of Bioinformatics • From Peptides to Carriers: Mapping Evolutionary Pathways • From Carriers to Active Transporters: The Bacterial Phosphotransferase System • From Active Transporters to Metabolons: The PTS-Glycolytic Complex

  3. INTRODUCTION The Power of Bioinformatics

  4. “Genomics has changed everything, but not our thought processes. We need a completely new way of thinking if man is to extract the information made available by genomics.” -Anonymous

  5. Bioinformatics • Every detail of every living organism is encoded within the genome of that organism. • It is the immense task of bioinformatics to decipher that information. • It is the even greater task of biosystematics to render that information intelligible to the human brain.

  6. Evolutionary Perspective • All of biology makes sense only in the light of evolution. • Any biosystematic approach to the classification of biological entities must take cognizance of evolution. • Molecular phylogeny reflects the evolutionary process and is therefore the most reliable guide to structure, function, mechanism, metabolism and physiology.

  7. Functional Structural Regulatory Sequence Physiological Input: Types of Data Bioinformatics and Biosystematics Pressures for and Origins of MDR Output: Types of Questions Answered Pathways of Transporter Evolution Intracellular (subcellular) Distribution Organism-specific Characteristics Horizontal Transfer between Organismal Kingdoms Independent Origins of Distinct Families Shuffling of Constituents between Multicomponent Transporters Bioinformatic approaches to answering fundamental questions about transport proteins

  8. +4 4 8 x2 +2 +4 6 2 x2 x2 12 24 FROM PEPTIDES TO CARRIERS Mapping Evolutionary Pathways

  9. Peptide Channels Protein Channels Carriers 1° Active Transporters Group Translocators

  10. A. Channel (1.A+1.C+1.E) # Topological Types 3 6 9 12 15 B. Carriers (2.A) 3 6 9 12 15 # TMSs/polypeptide chain

  11. Transporter Evolution: From 2 TMSs

  12. Proposed Common Origin for CRAC channels and CDF carriers Primordial hairpin (2TMSs) 2 X 2 2 X 3 not likely - 2 likely CDF (6TMSs) (Me2+:H+ Antiporters) Orai (4TMSs) (CRAC Ca2+ Channels)

  13. Transporter Evolution: From 3 TMSs

  14. Transporter Evolution: From 4 TMSs

  15. A. ABC1: 6TMSs Independent Origins for Three Families of ABC Porters B. ABC2: 6TMSs C. ABC3: 8TMSs

  16. ABC3 Topological Types A B

  17. Transporter Evolution: From 5 or 6 TMSs

  18. Transporter Evolution: From 10 or 12 TMSs

  19. Transporter Evolution: Variations on the 12 TMS Theme

  20. VIC +4 4 8 (2, 4, 6, 8, 12, 24) x2 +2 +4 6 2 x2 x2 12 24 MFS (6, 12, 14, 24) +1 x2 x2 +2 3 6 12 14 2 x2 24 APC 10 (10, 11, 12, 14) -2 -1 x2 6 11 12 +2 14 DMT (4, 5, 10, 30) +1 x2 x3 4 5 30 10 -1 MOP 10 (10, 12, (13?), 14, 15) -2 +1 +2 x2 6 14 15 12 Superfamily (Number of TMSs in Current Homologues) Proposed Pathway

  21. S S C S B A H S-P PEP I Pyruvate FROM CARRIERS TO GROUP TRANSLOCATORS The Bacterial Phosphotransferase System

  22. The PTS: Functional Complexity • Chemoreception • Transport • Sugar phosphorylation • Protein phosphorylation • Regulation of non-PTS transport • Regulation of carbon metabolism • Coordination of nitrogen and carbon metabolism • Regulation of gene expression • Regulation of pathogenesis • Regulation of cell physiology

  23. PTS: Structural Complexity IIC:The permease and receptor (sugar specific) IIB:The direct phosphoryl donor (permease specific) IIA:The indirect phosphoryl donor (family specific) EI and HPr:The general energy-coupling proteins(PTS pathway specific) Enolase:The energy-yielding enzyme PGI:The downstream substrate-converting enzyme Glycolysis:The interconnecting cyclic pathway ---------------------------------------------- PTS + Glycolysis:A metabolite-induced metabolon?

  24. Families of PTS Enzyme II Complexes PTS enzyme II complexes comprise of at least four (super)families that evolved independently of each other. • The Glc-Fru-Lac superfamily • The Asc-Gat superfamily • The Man family • The Dha family

  25. Proposed Origins of PTS Permeases (IICs) Glc-Fru-Lac superfamily (8 TMSs) Arose independently of other PTS permeases. Asc-Gat superfamily (12 TMSs) Arose from a 12 TMS permease. Man family (6 TMSs) May have arisen from a 6 TMS permease. Dha family (0 TMSs) Arose from a soluble Dha kinase.

  26. Mtl Fru Glc Glc’d Lac Chb The Glc-Fru-Lac Superfamily Fru:The original PTS Proposed Evolutionary Pathway: Mosaic origins of IIAs and IIBs: IIAGlc is not homologous to IIAMtl or IIANtr IIBGlc is not homologous to IIBChb Conclusion: PTS permeases arose by superimposition of diverse energy coupling proteins onto pre-existing permeases.

  27. The Asc-Gat Superfamily IICAsc homologues are often fused to IIA and IIB homologues, but IICGat homologues never are. IICAsc homologues are always encoded by genes in operons with IIA and IIB genes, but IICGat homologues can be encoded in operons lacking IIA and IIB genes. Some IICGat homologues are found in organisms that lack all other PTS proteins. Asc and Gat IIA and IIB constituents are distantly related to IIA and IIB constituents of the Glc-Fru-Lac superfamily. Conclusions: Asc permeases probably function exclusively via the PTS, but Gat homologues may retain secondary carrier function.

  28. The Man Family All constituents (IIA, IIB, IIC, and IID) differ structurally from all other PTS permease proteins. All members, but only members of this family, have IID constituents. The IIB constituents of the Man family are phosphorylated on His rather than Cys, but all others are phosphorylated on Cys. Conclusion: All constituents of the Man family arose independently of those of the other sugar-transporting PTS families.

  29. The Dha Family DhaK and DhaL correspond to the N- and C-termini of ATP-dependent DHA kinases. DhaM consists of three domains: IIAMan-DPr-ID. The three domains of DhaM are phosphorylated by PEP, EI and HPr, but DhaK and L are not phosphorylated. DhaK binds DHA covalently to a His residue and transfers the phosphoryl group from IIA of DhaM to tightly bound ADP in DhaL, and then to DHA. Thus DhaL is IIB; DhaK is IIC. Conclusion: PTS Dha non-permeases arose from soluble DHA kinases independently of all PTS permeases.

  30. FROM ACTIVE TRANSPORTERS TO METABOLONS The PTS-Glycolytic Complex

  31. S S + -------------- n II-PLx -------------- -------------- IIn-PLy -------------- -------------- IIn-PLy -------------- PTS enzymes -------------- IIn-PLy -------------- PTS enzymes Glycolytic enzymes Stabilized PTS Complex Free lateral diffusion Ligand binding +/- PL PTS energy- coupling enzyme association Glycolytic enzyme assembly S Complete Glycolytic Metabolon S Proteolipid Complex Proposed Steps in PTS Metabolon Construction

  32. Evidence for a PTS-Glycolytic Metabolon • IICs are complexed with PTS energy-coupling enzymes in E. coli cells, but are easily disrupted. (Saier et al., 1982. J. Cell Biochem. 18:231-238) • Stable PTS enzyme complexes are found in other bacteria. (Saier and Staley, 1977. J. Bacteriol. 131:716-718) • In E. coli the glycolytic pathway has been isolated as an equimolar multi-enzyme complex (1.65 MDa) exhibiting substrate compartmentation.(Mowbray & Moses, 1976. Eur. J. Biochem. 66:25-36) Benefits: Co-localization of PTS & glycolytic enzymes could provide high local PEP concentrations and allow substrate channeling.

  33. Glc ---------------- IIGlc~P ---------------- Glc-P ------------ IIGlc~P ------------ + Mlc Repression of ptsG ptsHI manXYZ ---------------- IIGlc ---------------- Mlc + Glc-P Activation of ptsG ptsHI manXYZ Glc Autoregulation of PTS Gene Expression (Plumbridge, 2002. Curr. Opin. Microbiol. 5:187-93)

  34. Peptide Channels Protein Channels Carriers 1° Active Transporters Group Translocators Multi-component Metabolons CONCLUSIONSFrom Peptides to Multi-component Metabolons

  35. Saier Lab (2005-2013) Erin Kim: Protein motif analysis. Se Kim: Transporter type comparisons. Richie Kimball: DsbB/D families. Graciela Lorca: CcpB and gene regulation in Bacillus; LAB genome sequencing & analysis. Qinhong Ma: Protein secretion. Thai Nguyen: Lab manager. Toff Peabody: Type II protein secretion. Chris Pivetti: Mechanosensitive channels. Shraddha Prakash: IT superfamily. Torston von Rozycki: Genomics; Transport protein fusion analysis. Soumya Singhi: Hardware maintenance; Software development. Aaron Stonestrom: HPr kinases; Genomics. Can Tran: TCDB; Software development; Transporter fusion protein analyses. Brit Winnen: TTT family; Genomics. Ming-Ren Yen: Transporter bioinformatics; MDR pump structure; PTS transport. Yufeng Zhai: Software development. Zhongge Zhang: PTS ascorbate transporter; MDR (EmrE) molecular genetics. Xiaofeng Zhou: Software development. Mohammed Aboulwafa: PTS biochemistry. Ravi Barabote: PTS bioinformatics; E. coli transcriptome analysis. Wolfgang Busch: Transporter classification. Thien Cao: General protein secretory (Sec) pathway. Claudia Chagneau: Biofilm formation in Bacillus. Abe Chang: Superfamily construction; Orthology software development. Soo-Keun Choi: Interregulon interactions in Bacillus subtilis. Yong Joon Chung: MDR characterization; Bacillus transcriptome; Transporter bioinformatics. Jeremy Felce: Genomics; Transport protein fusion analyses. Claudio Gonzalez: Treponema PTS. Guillermo Gosset: Transcriptome analyses in E. coli . Edgar Harvat: Fatty acid transport. Rikki Hvorup: Asc/Gat PTS superfamily; MOP superfamily. Mirium Khwaja: ABC exporter bioinformatics.

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