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

From Carriers to Active Transporters:

The Bacterial Phosphotransferase System

From Active Transporters to Metabolons:

The PTS-Glycolytic Complex

INTRODUCTION

The Power of Bioinformatics

“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

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.

Bioinformatics

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.

Evolutionary Perspective

Organism-specificCharacteristicsShuffling of

Constituentsbetween MulticomponentTransporters

Output:Types of QuestionsAnswered

HorizontalTransferbetweenOrganismal Kingdoms

IndependentOrigins of Distinct Families

Pathways of TransporterEvolution

Pressures for and Origins of MDR

Intracellular (subcellular) Distribution

Bioinformaticsand

Biosystematics

Sequence

StructuralFunctional

Regulatory

Physiological

Input:Types of Data

Bioinformatic approaches to answering fundamental questions about transport proteins

FROM PEPTIDES TO CARRIERS

Mapping Evolutionary PathwaysMapping Evolutionary Pathways

2

4

6x2

+4

8

12 24

+4

+2x2 x2

ProteinChannels

Carriers

1° ActiveTransporters

GroupTranslocators

Peptide Channels

# T

opol

ogic

al T

ypes

# TMSs/polypeptide chain

3 6 9 12 15

3 6 9 12 15

A. Channel (1.A+1.C+1.E)

B. Carriers (2.A)

Event Path Family

2 + 1 2 3 MIT; MFS

2 x 2 2 4 VIC; F-ATPase; MTB;

YiaAB; Connexin (?)

2 x 3 2 6 MC; F-ATPase; ABC1; YedZ; CDF; CRAC (-2)

2 + 4 2 6 VIC

2 + 6 2 8 VIC

Transporter Evolution: From 2 TMSs

2 X 2 2 X 3

Primordial hairpin (2TMSs)

Orai (4TMSs)(CRAC Ca2+ Channels)

CDF (6TMSs)(Me2+:H+ Antiporters)

not likely

likely

- 2

Proposed Common Origin for CRAC channels and CDF carriers

Transporter Evolution: From 3 TMSs

Event Path Family

3 + 1 3 4 Mot/Exb

3 x 2 3 6 MIP; DsbD; ABC2

3 x 2 3 7 LCT; Brho

3 + n-3 3 n MscS

Transporter Evolution: From 4 TMSs

Event Path Family

4 + 1 4 5 DMT

4 x 2 4 8 MTB; PNaS; LIV-E; ABC3

4 x 2 4 10 ABC3

4 x 3 (?) 4 12 LIV-E (?)

4 x 4

(4 x 2 x 2)

4 16 OPT

ABC1: 6TMSs

ABC2: 6TMSs

ABC3: 8TMSs

A.

C.

B.

Independent Origins for Three Families of ABC Porters

ABC3 Topological Types

A

B

Transporter Evolution: From 5 or 6 TMSs

Event Path Family

5 x 2 5 10 DMT; CaCA; ThrE; UT

5 x 2 5 11 MgtE

6 x 2 6 12 MFS; RND; PET; Chr; MOP; VIC; ArsB; ABC

6 x 3 6 16 H+-PPase

6 x 2 6 24(6 x 2 x 2)

VIC

Transporter Evolution: From 10 or 12 TMSs

Event Path Family

10 x 2 10 20 UT

10 x 3 10 30 DMT

12 x 2 12 24 MFS; VIC

Transporter Evolution: Variations on the 12 TMS Theme

Event Path Family

12 - 2 12 10 Chr; MOP; APC

12 - 1 12 11 AAAP; HAAAP; LIV-E(?)

12' 12 12' PIT

12 + 1, 2, 3 12 13-15 MOP

12 + 2 12 14 MFS; APC

Superfamily(Number of TMSs in Current Homologues)

Proposed Pathway

VIC(2, 4, 6, 8, 12, 24)

2

4

6x2

+4

8

12 24

+4

+2x2 x2

MFS(6, 12, 14, 24) 2 3+1 6 12x2 x2 +2 14

24

x2

APC(10, 11, 12, 14)

-16 12x2 -211+2

10

14

DMT(4, 5, 10, 30)

+1 x2 x34-1

5 10 30

MOP(10, 12, (13?), 14, 15)

+26 12x2 -210

14 +1 15

FROM CARRIERS TO GROUP TRANSLOCATORS

The Bacterial Phosphotransferase System

S-P

C

A

I

H

B

PEP

Pyruvate

S

S

S

The PTS: Functional Complexity

1. Chemoreception

2. Transport

3. Sugar phosphorylation

4. Protein phosphorylation

5. Regulation of non-PTS transport

6. Regulation of carbon metabolism

7. Coordination of nitrogen and carbon metabolism

8. Regulation of gene expression

9. Regulation of pathogenesis

10. Regulation of cell physiology

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?

Families of PTS Enzyme II Complexes

PTS enzyme II complexes comprise of at least

four (super)families that evolved

independently of each other.

1. The Glc-Fru-Lac superfamily

2. The Asc-Gat superfamily

3. The Man family

4. The Dha family

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.

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.

The Glc-Fru-Lac Superfamily

Mtl

Glc

Glc’d

Lac

Chb

Fru

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.

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.

The Dha FamilyDhaK and DhaL correspond to the N- and C-termini of

ATP-dependent DHA kinases.

DhaM consists of three domains: IIAMan-DPr-I

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.

FROM ACTIVE TRANSPORTERS TO METABOLONS

The PTS-Glycolytic Complex

Proposed Steps in PTS Metabolon Construction

StabilizedPTS

Complex

CompleteGlycolyticMetabolon

S

S--------------

IIn-PLy

--------------PTS enzymes

--------------IIn-PLy

--------------PTS enzymes

Glycolyticenzymes

Glycolyticenzyme assembly

Free lateral diffusion

ProteolipidComplex

S+

--------------n II-PLx

--------------

--------------

IIn-PLy

--------------

S

Ligandbinding

+/- PL

PTS energy-coupling enzyme

association

Evidence for a PTS-Glycolytic Metabolon

1. 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)

2. Stable PTS enzyme complexes are found in other bacteria. (Saier and Staley, 1977. J. Bacteriol. 131:716-718)

3. 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.

Autoregulation of PTS Gene Expression

Glc----------------

IIGlc~P----------------

Glc-P

Glc------------IIGlc~P

------------ +

Mlc

Repression of ptsGptsHI

manXYZ

----------------IIGlc

----------------Mlc

+ Glc-P

Activation of ptsGptsHI

manXYZ

(Plumbridge, 2002. Curr. Opin. Microbiol. 5:187-93)(Plumbridge, 2002. Curr. Opin. Microbiol. 5:187-93)

CONCLUSIONS

From Peptides to Multi-component Metabolons

1° ActiveTransporters Group

Translocators

Protein Channels

PeptideChannels

Carriers

Multi-componentMetabolons

Saier Lab (2005-2013)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.

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.