(Received for publication, April 24, 1995; and in revised form, July 10, 1995)
From the
The -hemolysin (
HL) polypeptide is secreted by Staphylococcus aureus as a water-soluble monomer that
assembles into lipid bilayers to form cylindrical heptameric pores
1-2 nm in effective internal diameter. We have individually
replaced each charged residue (79 of 293 amino acids) and four neutral
residues in
HL with cysteine, which is not found in the wild-type
protein. The properties of these mutants have been examined before and
after modification with the 450-Da dianionic sulfhydryl reagent
4-acetamido-4`-((iodoacetyl)amino) stilbene-2,2`-disulfonate (IASD).
This modification was highly informative as 28 of 83 modified
polypeptides showed substantially reduced pore forming activity on
rabbit erythrocytes (rRBC), while only five of the unmodified cysteine
mutants were markedly affected. Through detailed examination of the
phenotypes of the mutant and modified hemolysins, we have pinpointed
residues and regions in the
HL polypeptide chain that are
important for binding to rRBC, oligomer formation and pore activity.
Residues in both the N-terminal (Arg-66 and Glu-70) and C-terminal
(Arg-200, Asp-254, Asp-255, and Asp-276) thirds of the protein are
implicated in binding to cells. The His-35 replacement mutant modified
with IASD was the only polypeptide in this study that failed to form
SDS-resistant oligomers on rRBC. Altered hemolysins that formed
oligomers but failed to lyse rRBC represented the most common defect.
These alterations were clustered in the central glycine-rich loop,
which has previously been implicated as a component of the lumen of the
membrane-spanning channel, and in the regions flanking the loop.
Alterations in mutant and modified hemolysins with the same defect were
also scattered between the N terminus and His-48, in keeping with
previous suggestions that an N-terminal segment and the central loop
cooperate in the final step of pore assembly.
-Hemolysin (
-toxin,
HL) (
)is secreted
by Staphylococcus aureus as a water-soluble monomer, which
binds to membranes and forms oligomeric cylindrical pores(1) .
The 293-amino-acid polypeptide provides a useful model for examining
the structural changes that occur when a water-soluble protein
assembles into a lipid bilayer(2) . In addition,
HL has
been used as a tool in cell biology for the permeabilization of
mammalian cells(3) .
HL may also have applications in
biotechnology, e.g. in drug delivery or as a component of
biosensors(4) . Further,
HL contributes to the
pathogenicity of S. aureus in certain diseases(1) .
A working scheme for the assembly of HL summarizes an
accumulation of experimental data ().
STRUCTURES1-4
Rabbit erythrocytes (rRBC) carry an unidentified receptor for
HL that facilitates pore formation(5, 6) . The
receptor is not required for assembly as
HL can form pores in
protein-free bilayers(7, 8) . Early studies revealed
that the pore is an oligomer (e.g. Refs. 9, 10). More recent
studies by gel-shift electrophoresis and x-ray crystallography have
shown that the pore is a heptamer(11) . Based on their circular
dichroism, both the water-soluble monomer and the assembled pore are
largely
-sheet(10, 12) . Conformational analysis
by limited proteolysis has suggested that the
HL monomer consists
of two regions of approximately equal mass connected by a central
glycine-rich loop that encompasses residues
119-143(10, 13) . The loop becomes resistant to
proteolysis when
HL binds to membranes (10, 14) and, in the fully assembled pore, it is
likely to penetrate the bilayer, as evidenced by fluorescence
spectroscopy with derivatized single-cysteine mutants(15) , and
to line a segment of the lumen of the transmembrane channel, as
evidenced by the introduction of a metal ion-binding site that is
accessible from both sides of the membrane (16) . Assembly
intermediates have been demonstrated by the availability of mutants
that accumulate as membrane-bound monomers () or as
nonlytic oligomers ()(14, 17, 18) . The nonlytic
``prepore'' 3 is also a heptamer (18) .
Because mutations in both the N terminus and the central loop can yield
molecules that arrest as nonlytic heptamers(18, 19) ,
it seems likely that the N terminus and the loop cooperate in the final
step of assembly: conversion of the heptameric prepore to the fully
assembled pore (
). The fully
assembled pore () might contain a 14-stranded
barrel
formed by the loop by analogy with the porins, which contain 16- or
18-stranded barrels(20, 21, 22) . Such a
barrel could accomodate (23) the essentially nonselective
channel of 1- 2 nm internal diameter that is formed by
HL(7, 9) .
Here, we use scanning mutagenesis
and targeted chemical modification of HL to correlate sequence
with function. Residues and regions of the polypeptide that are
important for binding to rRBC, heptamer formation, and pore activity
are delineated. The study also reveals the residues in
HL that
will be available for chemical modification in investigations designed
to produce pores with novel properties.
Each cysteine mutant was
obtained in S-labeled form by in vitro transcription and translation (IVTT). Half of the sample was
modified with IASD, while the other half was subjected to a mock
reaction, which had little effect on the properties of the hemolysins.
Portions of the modified and unmodified
HL were examined by
SDS-PAGE to ensure that similar concentrations of each mutant were
obtained by IVTT (they were), to measure the gel shift produced by the
IASD modification(19) , to determine the extent of modification
(a few mutants were incompletely modified, see below), and to ensure
that the modification was confined to the single cysteine. The reaction
conditions (20 mM IASD, 2 h, room temperature, pH 8.5) were
the most extreme that can be used without significant derivatization of
additional functional groups on
HL. Prolonged modification gives a
diffuse band on SDS-PAGE, rather than a clean shift in mobility,
presumably because several additional nucleophiles (e.g. the
-amino groups of lysine) are slowly derivatized. An example of the
analysis is displayed (Fig. 1).
Figure 1:
Modification of S-labeled single-cysteine mutants of
HL with IASD
demonstrated by gel shift electrophoresis. WT-
HL (K8A) and a
selection of single-cysteine mutants were treated with the
sulfhydryl-directed reagent IASD as described in the text.
Unmodified(-) and modified (+) polypeptides were separated
by extended SDS-PAGE. An autoradiogram of the appropriate section of a
dried gel is shown. Lane 1, K8A (-); lane 2,
K8A (+); lane 3, R66C(-); lane 4, R66C
(+); lane 5, E70C(-); lane 6, E70C
(+); lane 7, K110C(-); lane 8, K110C
(+); lane 9, D152C(-); lane 10, D152C
(+); lane 11, R200C(-); lane 12, R200C
(+); lane 13, D254C(-); lane 14, D254C
(+); lane 15, E287C(-); lane 16, E287C
(+).
Additional portions of the
mutant and modified HL polypeptides were used to measure pore
forming activity by determining the rate of lysis of rRBC in a
quantitative microtiter assay. An example of such an experiment is
shown that exemplifies a variety of mutant phenotypes (Fig. 2).
Further portions of the polypeptides were incubated with rRBC at 20
°C for 1 h. SDS-PAGE was then used to determine the extent of
binding to the cells, the extent of oligomer formation, and the heat
stability of the oligomers. An example of this analysis is displayed (Fig. 3) for the same set of mutants shown in Fig. 2.
Figure 2:
Hemolytic activity of WT-HL and
unmodified and IASD-modified single-cysteine mutants of
HL. Traces
from an automated plate reader are shown. The wells contained
unmodified(-) and IASD-modified (+) polypeptides: row
1, K8A (WT)(-); row 2, K8A (WT) (+); row
3, R66C(-); row 4, R66C (+); row 5,
E70C(-); row 6, E70C (+); row 7,
K110C(-); row 8, K110C (+); row 9,
D152C(-); row 10, D152C (+); row 11,
R200C(-); row 12, R200C (+); row 13,
D254C(-); row 14, D254C (+); row 15, E287C
(-); row 16, E287C (+). Columns 1-8 contained 2-fold serial dilutions of the polypeptides. In each
window, the optical density at 595 nm recorded over 1 h is shown. The
top of the window represents 1.00 OD units. rRBC exhibit a large
decrease in light scattering upon lysis.
Figure 3:
Binding of WT-HL and unmodified and
IASD-modified single-cysteine mutants of
HL to rRBC,
oligomerization, and temperature stability of the oligomers as
determined by SDS-PAGE. Unmodified(-) and IASD-modified (+)
S-labeled
HL polypeptides were allowed to assemble on
rRBC. SDS-stable oligomers were detected by autoradiography after
separation by SDS-PAGE. A, samples heated to 50 °C before
electrophoresis. B, samples heated to 80 °C before
electrophoresis. Key: lane 1, K8A (WT)(-); lane
2, K8A (WT) (+); lane 3, R66C(-); lane
4, R66C (+); lane 5, E70C(-); lane 6,
E70C (+); lane 7, K110C(-); lane 8, K110C
(+); lane 9, D152C(-); lane 10, D152C
(+); lane 11, R200C(-); lane 12, R200C
(+); lane 13, D254C (-); lane 14, D254C
(+); lane 15, E287C(-); lane 16, E287C
(+). The diffuse band in lane 14 is thought to be
nonspecifically modified D254C; compare Fig. 1, lane
14. Radiolabeled markers denoted on the left side of the
autoradiogram were
C-methylated proteins (Life
Technologies, Inc.): myosin heavy chain (M
200,000); phosphorylase b (M
97,400); bovine serum albumin (M
68,000);
ovalbumin (M
43,000); carbonic anhydrase (M
29,000);
-lactoglobulin (M
18,400); lysozyme (M
14,300).
,
HL oligomers;
,
HL
monomers.
The results of measurements on all 166 altered hemolysins are
compiled in Table 1, which can be perused in conjunction with the
primary sequence of HL (Fig. 4). The results are also
summarized graphically to emphasize the distribution through the length
of the polypeptide chain of residues involved in the major functions of
HL: cell binding, oligomerization, and pore formation (Fig. 5, A and B).
Figure 4:
Amino acid sequence of wild-type HL (13) with minor amendments(1, 26) . Most of
the mutants described here were derived from
HL-K8A, a
protease-resistant mutant of
HL with the replacement Lys-8
Ala.
Figure 5:
Graphic
representations of the phenotypes of the altered hemolysins. A, chart displaying all altered hemolysins with properties
that differ greatly from WT-HL. 83 of 293 positions in
HL
were tested by individual substitution with cysteine as shown at the
bottom of the chart. Defects in the cysteine mutants were noted before
and after modification with IASD. Full details of the properties of the
mutants can be found in Table 1. For inclusion here, a defective
hemolysin has the following properties (see Table 1): lysis,
+ or below; binding, + or below; oligomerization, no
oligomers at 50 °C. Key. Unmodified mutants, defective in: rRBC
lysis,
; rRBC binding,
; oligomerization (no entry).
IASD-modified mutants, defective in: rRBC lysis,
; rRBC binding,
; oligomerization,
. B, functional properties of
HL assigned to regions of the primary sequence. The assignments
are consistent with the present work and previous findings.
Nevertheless, in the absence of structural information, they are likely
to be oversimplified. The sequence in B is aligned with the
chart in A and is given in single-letter code in Fig. 4.
As expected, the properties of the IASD-derivatized mutants are more dramatically affected than the properties of the cysteine mutants themselves. Twenty-eight showed greatly reduced pore forming activity. Few cysteine mutants in the central one-third of the polypeptide and none in the glycine-rich loop itself remain active after treatment with IASD. Residues affected by IASD attachment are also scattered through the most N-terminal 50 amino acids, but there are also many unaffected residues in this region. Again, the most common phenotype is arrest as a nonlytic oligomer, presumably similar to the assembly intermediate .
The purpose of this study was to apply cysteine scanning
mutagenesis to assign functions to sequences in the HL polypeptide
chain. Most of the single-cysteine mutants were highly active, as noted
in other cases, e.g. the lactose permease of E.
coli(33) . Therefore, the value of the mutagenesis was
enhanced by using targeted chemical modification of cysteine to produce
a set of more drastically modified hemolysins corresponding to the
single-cysteine mutants. The assignment of function after point
mutagenesis is limited by the interdependence of sites that are far
apart in the primary sequence of a polypeptide (an issue addressed in
the accompanying paper(34) ). Nevertheless, the study has
provided valuable insights into the function of a pore-forming protein.
Previously, we divided the HL chain of 293 amino acids into
three functional regions: the N- and C-terminal domains and the
glycine-rich central loop. The present findings appear to vindicate
this approach, although it seems likely that the borders of the central
region should be expanded and that the N-terminal domain can be further
subdivided.
Here, we find that mutants of HL with
single cysteines located near the center of the glycine-rich loop
(positions 127-131) are highly active and that all of them are
largely inactivated by IASD modification. Perhaps the mass and charge
of the stilbene disulfonate prevents membrane penetration by the
altered loop and subsequent transmembrane channel formation (
). It is noteworthy that the central
loop is flanked on one side by Lys-110 and on the other by Asp-152, two
of only four residues that yield unmodified cysteine mutants in which
pore formation, but not oligomerization, is prevented. The region
immediately upstream from the central loop is sensitive to IASD
modification throughout, suggesting that the channel forming apparatus
may extend to around residue 100. IASD sensitivity is also found
C-terminal of the loop in the region encompassing amino acids 147 to
168, although insensitive residues are also found here.
Interestingly, residues 169-197 have a similar character to
the glycine-rich loop. An extended stretch of neutral residues is
broken at its midpoint by three charged residues (Asp-183, Arg-184, and
Asp-185). Single-cysteine mutants at these positions are active and
pore formation, but not oligomerization, is prevented after reaction
with IASD, as observed for Asp-127, Asp-128, and Lys-131 in the central
loop. Therefore, it is conceivable that residues 169-197
constitute a second membrane-penetrating region. Indeed, in the central
one-third of the protein, there is an imperfect but suggestive mirror
symmetry in the properties of residues on either side of the midpoint
of the polypeptide chain (Fig. 5A). It should be worth
examining the extended regions of uncharged sequences in HL in
more detail by the present approach and with fluorescent probes that
respond to hydrophobic environments(15) .
The present findings are in keeping with the earlier studies. Near
the N terminus, at positions 24 and 35, we find two residues of the
four in HL that prevent pore formation when changed to cysteine
(the fifth inactive cysteine mutant, E70C, is defective in binding to
rRBC). Sites that are sensitive to IASD are also scattered throughout
the first 50 amino acids, although there are also many insensitive
sites.
The second half of the N-terminal one-third of HL
responds quite differently to mutagenesis. When altered, most residues
between 50 and 100 do not yield defective
HL molecules except for
residues 66 and 70, which appear to be involved in binding to rRBC.
From a pragmatic viewpoint, the study has
revealed residues that may be chemically modified in attempts to
produce pores that have novel properties. Indeed, after this work was
complete, the data were used to guide the construction of a
photoactivatable HL by targeted chemical modification of
cysteine(37) . Cysteine residues might also be used in the
construction of hybrid proteins or for attaching the pores to surfaces.
The discovery of heat stable oligomers is also interesting in the
context of biotechnology(38) , although in the present study
they are largely associated with loss of activity.