CHAPTER 8 – INTRODUCTION TO BACTERIAL ABC PROTEINS

CHAPTER 8 – INTRODUCTION TO BACTERIAL ABC PROTEINS CHAPTER 8 – INTRODUCTION TO BACTERIAL ABC PROTEINS CHAPTER 8 – INTRODUCTION TO BACTERIAL ABC PROTEINS CHAPTER 8 – INTRODUCTION TO BACTERIAL ABC PROTEINS CHAPTER 8 – INTRODUCTION TO BACTERIAL ABC PROTEINS CHAPTER 8 – INTRODUCTION TO BACTERIAL ABC PROTEINS CHAPTER 8 – INTRODUCTION TO BACTERIAL ABC PROTEINS

In Chapter 1 in this volume, E Dassa has

reviewed the classification of ABC proteins,

including prokaryote representatives and their

transport substrates in the many cases where

these have been identified Previous general

reviews have also discussed the ABC proteins

in Escherichia coli (Linton and Higgins, 1998),

Bacillus subtilis (Quentin et al., 1999) and

Mycobacterium tuberculosis (Braibant et al., 2000)

and more specifically concerning bacterial

ABC exporters in E coli (Fath and Kolter, 1993;

Young and Holland, 1999) The purpose of

this introductory chapter is therefore briefly to

highlight some of the major characteristics of

bacterial ABC systems and the breadth of their

functions

Prokaryote ABC-dependent transport systems,

whether exporters or importers, all adhere to

the usual formula of a basic four-unit structure,

two membrane components and two units of

ABC-ATPases The membrane components and

the ABCs may be identical or non-identical and

can be fused pairwise in different combinations

as shown in Chapter 1, although unlike those

commonly found in eukaryotes no examples

of all four subunits fused together have been

identified in prokaryotes In describing ABC-dependent transport systems, it is important to emphasize that the term ABC (ATP-binding

cassette, Hyde et al., 1990) is synonymous with

ABC-ATPase, whether present as a subdomain

or an independent polypeptide The term ABC transporter, on the other hand, describes the ABC-ATPase (also called a traffic ATPase; Ames and Lecar, 1992) plus its associated integral membrane domains, whether fused to the ABC

or separately encoded This core transporter or translocation complex may be further supple-mented with essential accessory or auxiliary subunits (usually encoded separately): the external ligand-binding protein in the case of ABC importers, or the MFP (membrane fusion protein) and the OMP-F (outer membrane pro-tein/factor) or OMA (outer membrane auxil-iary) integral to the inner membrane and outer membrane, respectively In the case of ABC transporters, the whole complex may some-times be referred to as the translocon, whilst for the importers, the term permease is also used to describe the entire complex

Whilst ATP is the substrate for the

ABC-ATPase, the molecule or ion being transported

by the ABC transporter is variously described

as a substrate or a transport substrate or an allocrite Since in our view, in the vast majority

of cases, the component being transported remains unmodified by the process, the term

‘substrate’ is inappropriate, and we prefer allo-crite, a term we coined, loosely derived from the Greek meaning a substance transported or

ABC Proteins: From Bacteria to Man ISBN 0-12-352551-9

Copyright 2003 Elsevier Science Ltd All rights of reproduction in any form reserved

8

I NTRODUCTION TO B ACTERIAL

ABC P ROTEINS

CHAPTER

exported (Blight and Holland, 1990; Young and

Holland, 1999)

Probably the earliest detailed studies of ABC

proteins were carried out in bacteria in the

1970s and 1980s, concerning the mechanism of

uptake of solutes such as histidine and maltose,

mediated by the ABC proteins, HisP (Ames and

Nikaido, 1978) and MalK (Bavoil et al., 1980) in

Salmonella typhimurium and E coli, respectively.

These proteins were initially recognized as

binding ATP and subsequently as energy

generators for transport (Hobson et al., 1984;

Shuman and Silhavy, 1981), through the

hydrol-ysis of ATP As seen in Chapter 9, although we

still have much to learn concerning the

mecha-nism of transport driven by, for example, HisP

and MalK, structural and genetic studies of the

importing ABCs continue to be the most

advanced

ABC-ATPases are now recognized as one of the major superfamilies of proteins, represented

in all three kingdoms of life and found in all

organisms so far analyzed ABC proteins are

particularly abundant in prokaryotes, with

genes constituting from close to 1% and up to

more than 3% amongst the 19 eubacteria and

6 archaea, respectively, surveyed in Chapter 1

The very recent sequence of the Agrobacterium

tumefaciens genome describes the highest

num-ber recorded so far, 153, excluding orphan ABCs

(with no discernible membrane domain

associ-ates) Thus, ABC transporters constitute 60% of

all transporters and about 3% of all predicted

polypeptides in the A tumefaciens genome

(Goodner et al., 2001; Wood et al., 2001) All the

known bacterial genomes, with one exception,

Treponema pallidum (only 1.14 Mb), encode all the

three main categories of ABC protein discussed

below, the exporters, orphans and importers

Curiously, T pallidum and four out of the six

archaeal genomes listed in Chapter 1, apparently

do not encode any exporters

In Chapter 1, based on cluster or phylogeny analysis of sequences constituting the ABC

polypeptides, over 600 examples out of the more

than 2000 entries in the current databases, 33

distinct clusters were identified These are assigned so far to three major classes, all strongly represented in bacteria Class 1 contains the large family of exporters Class 2 is a small family of orphans, with no known membrane protein associates and, at least in some cases, with no connection to membrane transport processes, for example the bacterial UvrA protein essential for specific DNA repair processes Class 3 is func-tionally probably a more heterogeneous family, since it probably contains both importers and exporters This heterogeneity may necessitate a future separation into at least two distinct classes

Importantly, an additional important group of ABC proteins present in both bacteria and eukaryotes, which are not involved in trans-port but concerned with DNA repair or recom-bination, have yet to be classified as class 1, 2

or 3 and may well constitute a completely new class Such an example, the ABC domain of

Rad50 from Pyrococcus furiosus, involved in

homologous recombination, has recently been crystallized and the structure determined

(Hopfner et al., 2000) The ABC domain

con-tains the two characteristic lobes or arms found

in HisP (Hung et al., 1998) This contains all the

expected, highly conserved motifs, the Walker

A, Q-loop, Walker B and the downstream histidine (Linton and Higgins, 1998), present

in Arm-I, the RecA-like, catalytic domain

(Geourjon et al., 2001) Similarly, Rad50

con-tains the signature motif in the smaller Arm-II, sometimes referred to as the helical (Ames and Lecar, 1992) or signaling/regulatory domain (Holland and Blight, 1999) In reality, in the intact Rad50 molecule, the helical or signaling domain is interrupted by the insertion into helix 3 of 600 residues forming a long coiled coil region, thereby separating the Walker A from the Walker B domain Interestingly, as discussed in Chapter 11, structural studies so far indicate that functionally different types

of ABC protein display the greatest variation in

structural organization in the helical domain,

frequently affecting helix 3

The extensive coiled coil region of Rad50, facilitating dimerization of these large

mol-ecules, restoring the close proximity of the

Walker A and B motifs for nucleotide binding,

is in fact diagnostic of a large family of bacterial

and eukaryote SMC (structural maintenance

of chromosome) proteins (Melby et al., 1998;

Soppa, 2001), many of which are involved in

condensation of DNA, including the SMC

pro-tein in B subtilis required for chromosomal

segregation (Graumann et al., 1998) Notably,

whereas Rad50 has a relatively well-conserved

LSGG motif compared with the ‘classical’ ABC

proteins, other SMCs have a more ‘degenerate’

version of this signature motif Finally, perhaps

the most distant relatives, but still considered

as ABC proteins (Aravind et al., 1999), are the

DNA repair enzymes such as the bacterial

MutS These proteins contain minimal Walker

A and B motifs and have the same overall fold

for the catalytic domain as HisP (Lamers et al.,

2000), but the signature motif is significantly

diverged from that of HisP, and indeed much

of the region equivalent to the helical domain

of HisP is absent (Geourjon et al., 2001).

Class 1 ABC-ATPases (fused to a membrane

domain), and apparently some class 3 proteins

(encoded independently from the membrane

domain), constitute at least eight distinct

fami-lies, all concerned with the export of a wide

range of compounds These include extremely

large polypeptides, greater than 400 kDa in

some cases (Chapter 11), polysaccharides, a

wide variety of antibiotics, many drugs

(Chapter 12), and certain lipids (Chapter 7) A

fascinating adaptation of the modular structure

of an ABC protein is shown in the ABC

compo-nent of the translocators for non-lantibiotics

secreted by Gram-positive bacteria In these

cases the N-terminal domain of the ABC

trans-porter carries a cytoplasmic extension to the

membrane domain (Havarstein et al., 1995),

which constitutes a cysteine protease, necessary

for processing the antibiotic peptide as it exits

from the cell (see Chapter 11) Some evidence

suggests that class 1 ABCs are also involved

in exporting fatty acids and Na⫹ions as

trans-port substrates or allocrites As reviewed in

Chapter 1, however, firm evidence for the

iden-tity of allocrites in many cases is still lacking

Importantly, whilst inferences regarding poten-tial allocrites for class 1 transporters can be drawn from cluster analysis through guilt by association with well-characterized transpor-ters, this approach is not necessarily reliable

One of the largest exporter families, DPL(see Chapter 1), contains at least 11 subfamilies of bacterial ABCs, which are involved in the export

of allocrites as diverse as lipids, large polypep-tides, or a wide range of drugs Of course, we cannot rule out the possibility that some of these transporters export in reality more than one type of compound, as has been demonstrated

for Pgp (Johnstone et al., 2000; Raymond et al.,

1992) As a further complication, the ABC trans-porters in the Prt and Hly clusters in the hetero-geneous DPL family require additional, specific auxiliary membrane proteins in order to com-plete, if not provide, the actual translocation pathway (Chapter 11)

Interestingly, from knowledge that is avail-able so far, the bacterial exporters appear to ful-fill a variety of important cellular functions, for example the secretion of factors required for dominating other bacterial species in the envi-ronment, for colonization of plant, insect or animal hosts leading to pathogenic infection or symbiosis, for the removal of toxic compounds and for the biogenesis of several constituents

of the organism’s own cellular envelope Many

of the latter are essential for respiratory func-tions, the integrity of the bilayer, simple surface protection and even movement of the bacteria

Moreover, some ABC exporters have been impli-cated in various developmental and differen-tiation programs, although their precise roles and allocrites transported in these cases are mostly obscure For further information and lit-erature sources on several of these aspects, see other chapters in Parts I and II in this volume

The class 2 group of ABC proteins are present in all organisms but are curious exceptions to the rule that the ABC proteins are always involved

in transport processes across membranes The functions of these proteins as a group are quite diverse and surprising, being involved in trans-lation of polypeptides, drug and antibiotic resist-ance, and in DNA repair, although only the latter two have been documented in bacteria so far (Chapter 1) It is intriguing to know what

common principles might govern the action of

a highly conserved ABC domain involved in

processes as different as membrane transport,

DNA repair and protein synthesis Interestingly,

in the bacterial UvrA protein, a tandemly

dupli-cated ABC, there is an insertion of a DNA

bind-ing motif, a zinc fbind-inger, between the Walker A

and the signature motif in each ABC domain

(see, for example, Husain et al., 1986; Yamamoto

et al., 1996) This insertion occurs in a position

close to the equivalent of the interface between

the two lobes in the HisP structure, which

pre-sumably must affect the regulation of UvrA

function As a further curiosity, if not a mystery,

ABCs in this group of class 2 orphans include

proteins, also with duplicated ABC domains,

from, for example, Staphylococcus aureus and

Streptomyces antibioticus (Mendez and Salas,

2001; Ross et al., 1995), responsible for resistance

(and immunity in some cases) to certain drugs

and antibiotics The simplest explanation would

be that these ABCs do work in conjunction with

some membrane protein to export the drugs but,

despite intensive efforts, such proteins have not

yet been identified

The class 3 ABC transporters in bacteria

consti-tute an enormous family of import systems for

small molecules The transport complex is

com-posed of two molecules of an independently

encoded ABC protein(s), a hetero- or homodimer

of integral membrane proteins constituting the

translocation pathway, and an external

ligand-binding protein, amongst which the most

characterized, the periplasmic binding proteins

in Gram-negative bacteria are considered in

Chapter 10 The class 3 importers have been

assigned to at least nine major families in the

phylogeny analysis in Chapter 1 The allocrites

transported cover a wide range of essential and

non-essential molecules, including several metal

ions, iron chelates, vitamin B12, mono-, di- and

oligosaccharides, polyols, polyamines, inorganic

anions such as sulfate, nitrate and phosphate,

phosphonates, peptide osmoprotectants and

ssother di- and oligopeptides From these

examples, the import systems for histidine and

maltose will be considered in some detail in

Chapter 9, and for uptake of osmoprotectants

in Chapter 13 As already indicated, despite the

range of allocrites transported in this very large

family, the nature of the different translocators is

surprisingly uniform: an external ligand-binding

protein, free in the periplasm in Gram-negative bacteria whilst it may be anchored to the mem-brane surface in Gram-positive bacteria; two membrane proteins for transport, carrying the EAA interaction motif; and a highly conserved ABC protein on the cytoplasmic side of the inner membrane Since evidence of exchangeability

of one ABC component for another in these otherwise very similar systems has been rarely indicated in the literature, we must assume that each ABC is tailormade for contact and intramolecular signaling with its cognate mem-brane domains

Recent studies of two ABC-dependent solute uptake systems responsible for transport of general amino acids and branched amino acids

in Rhizobium leguminosarum have revealed the

surprising finding that such systems can

appar-ently also export these amino acids Moreover,

the same phenomenon was demonstrated with

histidine transport in S typhimurium (Hosie

et al., 2001) This reverse transport or

bidirec-tional capacity of these ABC transporters raises some complex questions concerning the solute pathway in the two different directions In addi-tion, it is not yet clear whether ATPase activity

is required for the efflux process (P Poole, per-sonal comunication)

Whereas great progress has been made in the comparative, phylogenic analysis of the ABC domains, leading to prediction of possible func-tion in the absence of other evidence in many cases, the cluster analysis of membrane domains has lagged far behind This clearly hampers insights into the mechanistic role of these domains as potential translocation pathways and these are poorly understood Nevertheless,

as discussed in Chapter 9, the early recognition

(Dassa and Hofnung, 1985) of the EAA motif,

apparently completely conserved without exception within a cytoplasmic loop of the membrane components of all the bacterial ABC importers, has ultimately led to the identifica-tion of this as a specific point of contact with a region of the helical domain of the ABC-ATPase

This is presumably also a critical point in the

intramolecular signaling pathway, coordinating

transport and energy generation

Importantly, the EAA motif is not present in any of the exporters, indicating that during

evolution ABC-ATPases, in bacteria at least,

have associated with more than one type of

membrane domain Furthermore, the failure so

far to detect any kind of conserved motif in the

membrane domains of ABC exporters perhaps

emphasizes, in contrast to the importers, the

wide variation in both the mechanism and

the pathway of molecular signaling between

the membrane and ABC components of the

exporters As indicated below and discussed in

Chapter 7, the elucidation of the structure of

the membrane domain of the E coli MsbA

protein will now enormously stimulate this

aspect of ABC studies

Notably, some of the most advanced structural

studies of ABC transporters have come from

bac-terial import and, more recently, bacbac-terial export

systems Thus, we now have high-resolution

structures for ABC importers, HisP (Hung

et al., 1998), a MalK from Thermococcus litoralis

(Diederichs et al., 2000), one ABC in the family

of branched-chain amino acid transporters and

one of unknown function (Karpowich et al.,

2001; Yuan et al., 2001) In this laboratory, we

have recently obtained the high-resolution

structure of the ABC domain of HlyB (Schmitt

et al., in preparation), a member of the large

DPL family, which includes the mammalian

TAP and Pgp (Mdr1) proteins The implications

of all these structural advances will be

consid-ered in other chapters As discussed in Chapter

7, a very major and exciting advance in the

field was made by the presentation of the first

structural data at 4.5 Å for the intact bacterial

exporter MsbA from E coli (Chang and Roth,

2001) This provides the first sign of the nature

of the membrane domain, and, in particular,

that of the membrane-spanning domains

These are finally shown to be helices, settling

some previous controversies Most crucially, of

course, this overall structure of MsbA has

pro-found implications for at least a global

under-standing of how the action of the membrane

and ABC domains may be coordinated Chang and Roth (see also Higgins and Linton, 2001)

on the basis of this structure have already pro-posed an exciting solution to a long-standing puzzle – how close are the ABC domains in the transporter? – that most likely they are inter-faced at some point in the catalytic cycle (see also Chapter 6), but under the influence of the membrane domains they are well separated in the absence of any transport substrate Unfor-tunately, mechanistic studies of the nature of the catalytic cycle of ABC proteins in bacteria, and its relationship to the transport function, have lagged relatively far behind those for some of the mammalian proteins However, recent advances in purifying and reconstitut-ing proteins of the maltose and histidine uptake systems (see Chapter 9), combined with the power of microbial genetics, promise much for the future

Excitingly, as this volume goes to press the high-resolution structure of the bacterial ABC import system for vitamin B12, BtuCD, is

reported (Locher et al., Science 296, 1091–1098),

providing many new insights into the mecha-nism of ABC-dependent transport

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