The
E.
coli Porin Proteins
OMPF
and Maltoporin (LamB Porin)
Kristin Flammer
'99, Aaron Downs '00, and David Marcey
Contents:
I.
Background
II.
Porin Structure and Function: The General Diffusion Pores Matrix Porin
III.
Maltoporin Monomer Structure
IV.
Structural and Functional Relationships in maltoporin
V.
References
I. Background
The porin superfamily
contains a number of homotrimeric (transmembrane) proteins that form water-filled
pores across the outer cell membranes of gram-negative bacteria. It has
been possible to isolate a number porins in their active forms and generate
monomer crystal structures.
Porins allow bacterial
cells to interact with their environment through the passive diffusion
of small (<600 Da) hydrophillic solutes across bacterial membranes.
Most porins form general, non-specific channels that are regulated by environmental
changes.
A few porins display substrate-specificity
in addition to general diffusion properties. Maltoporin, also known as
the LamB porin, is among the best studied examples of the substrate-specific
porins. It is responsible for the guided diffusion of maltose and maltodextrins
into E. coli cells. Maltoporin is also one of the many porins which
contains a bacteriophage recognition sequence. It was recognized as the
E.
coli receptor for phage lambda before its role in the translocation
of sugar was elucidated.
II.
Introduction to Porin Structure and Function: The General Diffusion Pores
Matrix Porin (OMPF)
Although there is little
to no sequence homology among porins, they often share a strong structural
resemblance. OMPF (outer
membrane
protein
f) is among the most general E. coli porins and is, therefore,
used as a reference point in discussing the structures of more specific
porins such as maltoporin. The active form of the OMPF protein is formed
by the joining of three identical monomers. A major component of OMPF monomer
is a 16-stranded antiparallel beta sheet barrel
that encloses a channel 7 X 11 Å in diameter <
The
closed nature of the barrel keeps polar main chain atoms away from the
membrane
core by occupying them in interstrand H-bonds. Monomer barrels are
further stabilized by internal loop structures and a hydrogen bonding brace
created by Tyr residues on the barrel walls
<>.
The profound stability
of porin trimers is a product of tight monomer interactions. The monomer
interface contains the amino-carboxy salt bridge and provides a hydrophobic
core through extensive (mainly hydrophobic) residue interactions over 35%
of the molecule. The C termini and strand 16 are essential to trimer formation.
Points of contact include the barrel walls and peripheral contacts between
L1 and L5. Cell surface loop 2 <>
increases trimer stability by folding into the channel of an adjacent monomer
and hydrogen bonding with loops 2, 3, and 4. Salt bridges are also formed
with arginines in L3.
Many of the channel's
functional properties stem from its loops. Some loops
pack together to form a hydrophilic umbrella structure over the channel
opening <>.
It is inferred that these loops protect the channel and screen solutes
based upon size and charge.
Loop
3 folds into the channel and packs against the channel wall, forming
a 9 Å constriction zone halfway through the barrel <>.
Contributors to the constriction with the barrel wall are highly conserved
Pro,
Glu, Phe, Gly residues at the tip of loop 3 as well as Asp113
and Glu117 <>.
The constriction zone determines a pore's selectivity and absolute solute
size limitation. The channel diameter widens to 15 X 22 Å below the
constriction <>.
III. Maltoporin Monomer Structure
Although there is no recognized
sequence homology between maltoporin and OMPF, there are a number of folding
similarities. Maltoporin monomers
are 80 residues longer and take the form of 18-stranded
beta barrels <>
that enclose smaller channels, 5-6 Å in diameter. The barrel is completed
by a linking of the amino (Val1)
and carboxy (Trp421) termini in
strand 18 <>,
and barrel stability and active trimer formation are influenced by many
of the same types of residue interactions described for OMPF.
Successive stands are
connected though periplasmic beta hairpin turns and
irregular cell surface loops <>.
The loops found in maltoporin are considerably longer than those found
in OMPF. Parts of loop 6, together with loop
4, loop 5, and loop
9 form the protective compact structure over the channel entrance
<>.
In addition to loop
2 from an adjacent subunit <>,
three monomer surface loops fold into the channel <>.
As in OMPF, aromatic and ionizable residues in loop
3 form a mid-channel constriction zone with residues on the barrel
wall <>.
Additionally, residues from loop 1 and loop
6 narrow the channel entrance <>.
IV. Structure/Function Relationships in
Maltoporin
A.
Lambda Phage Receptor
An intact maltoporin trimer
is required for lambda phage infection of E. coli. The monomer
loops forming the protective umbrella structure
<>
are the most variable regions among porins. This variability may serve
to protect bacteria from infection. A number of surface
point mutations (residues 154, 155, 164, 259, 382, 386, 387, 394,
401) in these regions have been shown to have no effect on sugar translocation
while conferring lambda phage resistance <>.
Another series of resistance
mutations occurs in sites 151, 152, 163, 245, 247, and 250, all
buried by loop compaction <>.
These mutations may affect umbrella behavior or surface structure and have
variable effects on sugar translocation. An additional mutation
site (residue18) abolishes translocation and is located on loop
1 <>.
The precise nature and structure of the lambda binding site is unclear.
B.
Sugar Translocation
Sugar diffusion is influenced
by residues from loop 3 (constriction zone),
plus loop 1 and loop
6, which narrow the channel entrance <>,
as well as by residues on the barrel walls. A series of aromatic residues
(Tyr41, Tyr6,
Trp420,
Trp358,
Phe227)
forms a left-handed helical path down the channel lining <>.
Trp74
from an adjacent loop 2 adds to the top of
this path <>.
These aromatic residues are spaced 6-7 Å apart and may stack with
hydrophobic faces of sugar molecules, forming transient bonds to guide
the sugar from the channel opening through the constriction zone.
This aromatic helical
path, or "greasy slide", is surrounded by a number of ionizable
residues from the channel lining that are assumed to replace the
hydration shells of diffusing molecules and convey sugar specificity to
the channel <>.
Although the precise molecular
mechanisms underlying maltoporin function are still unknown, translocation
is currently modeled as follows. Residues from L1 and L6 serve to orient
sugar molecules which are then guided through the constriction zone along
the greasy slide. Channel specificity is determined by residues in the
channel lining.
V. References
Branden, C. and J. Tooze.
Introduction
to Protein Structure (Garland, New York, 1991).
Cowan, S. W., T. Schirmer,
G. Rummel, M. Steiert, R. Ghosh, R. A. Pauptit, J. N. Jansonius, and J.
P. Rosenbusch. (1992). Crystal structures explain functional properties
of two E. coli porins. Nature 358: 727-733.
Hofnug, M (1995). An Intelligent
Channel (and More). Science 267: 473-474.
Schirmer, Tilman, Thomas
A. Keller, Yan-Fei Wang, and Jurg P. Rosenbusch. (1995). Structural Basis
for Sugar Translocation Through Maltoporin Channels at 3.1 A Resolution.
Science
267: 512-514.
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