(Received for publication, April 24, 1995; and in revised form, July 10, 1995)
From the
-Hemolysin (
HL), a pore-forming polypeptide of 293
amino acids, is secreted by Staphylococcus aureus as a
water-soluble monomer. Residues that play key roles in the formation of
functional heptameric pores on rabbit red blood cells (rRBC) have been
identified previously by site-directed mutagenesis.
HL-H35N, in
which the histidine at position 35 of the wild-type sequence is
replaced with asparagine, is nonlytic and is arrested in assembly as a
heptameric prepore. In this study, second-site revertants of H35N that
have the ability to lyse rRBC were generated by error-prone PCR under
conditions designed to produce single base changes. The analysis of 22
revertants revealed new codons clustered predominantly in three
distinct regions of the H35N gene. One cluster includes amino acids
107-111 (four revertants) and another residues 144-155
(five revertants). These two clusters flank the central glycine-rich
loop of
HL, which previously has been implicated in formation of
the transmembrane channel, and encompass residues Lys-110 and Asp-152
that, like His-35, are crucial for lytic activity. The third cluster
lies in the region spanning amino acids 217-228 (eight
revertants), a region previously unexplored by mutagenesis. Single
revertants were found at amino acid positions 84 and 169. When compared
with H35N, the heptameric prepores formed by the revertants underwent
more rapid conversion to fully assembled pores, as determined by
conformational analysis by limited proteolysis. The rate of conversion
to the fully assembled pore was strongly correlated with hemolytic
activity. Previous work has suggested that the N terminus of
HL
and the central loop cooperate in the final step of assembly. The
present study suggests that the key N-terminal residue His-35 operates
in conjunction with residues flanking the loop and C-terminal residues
in the region 217-228. Hence, reversion mutagenesis extends the
linear analysis that has been provided by direct point mutagenesis.
-Hemolysin (
HL), (
)a polypeptide of 293
amino acids, is secreted by Staphylococcus aureus as a
water-soluble monomer and assembles to form a heptameric pore on pure
lipid bilayers or on biological membranes such as those of red blood
cells(1) . Based on biochemical, biophysical, and molecular
genetic studies, four stages in the assembly of
HL have been
defined (Refs. 2, 3, and the accompanying paper(4) ). Monomeric
HL in solution (Structure 1) comprises two domains connected by a
central glycine-rich loop (residues
119-143)(5, 6) .
HL first binds to the
membrane surface as a monomer (Structure 2). A nonlytic oligomer
consisting of seven subunits (3, 7) is then formed
through interactions in which the C-terminal domain may play a
predominant role (Structure 3)(2, 4) . The subunits
then further penetrate the membrane to form the heptameric lytic pore
(Structure 4). Recent evidence suggests that the central loop lines
part of the lumen of the transmembrane channel in the fully assembled
structure(8, 9) .
In vitro mutagenesis has
identified residues that are important for the membrane binding,
assembly, and pore forming activity of HL (summarized in (4) ). For example, replacement of His-35 by Leu, Ile, Ser,
Thr, Arg, Pro(10, 11) , Cys(12) , Asn, Trp, or
Gln (13) has been shown to eliminate or greatly reduce
hemolytic activity. However, when Cys-35 in H35C was alkylated with
iodoacetamide to form the modified residue S-carboxamidomethylcysteine, hemolytic activity was
restored(13) . Therefore, the volume of the residue at position
35, and perhaps other factors such as the polarity and hydrogen bonding
potential of the side chain, play a crucial role, while the ability of
the residue to ionize is unimportant(13) . Recently, four
additional amino acids, Asp-24, Glu-70, Lys-110, and Asp-152, have been
identified, which, when individually replaced with cysteine, yield
hemolysins with greatly reduced hemolytic activity(4) . Three
of these mutants (D24C, K110C, and D152C) form oligomers but not
functional pores, suggesting that like H35N (3) they are
arrested at the prepore stage of assembly (Structure 3). By contrast,
the loss of activity in the mutant E70C is attributed to its low
affinity for the rRBC membrane.
The aim of the present study was to produce second-site mutations that restore the activity of the H35N mutant, and thus to step beyond the linear analysis of point mutagenesis by obtaining information about the interactions between key residues in different domains of the polypeptide chain.
A portion of each IVTT mix was used to compare the
hemolytic activity of the revertants. In a quantitative assay, the 19
mutants showed a wide range of activities (Table 1). As expected
from the screening procedure, all were more active than H35N and a few
had activity comparable with that of WT-HL.
Figure 1:
Binding of WT-HL,
HL-H35N and
the revertants of H35N to rRBC and subsequent oligomerization. In
vitro translated
HL polypeptides, radiolabeled with
S, were allowed to bind to rRBC for 1 h at 20 °C.
Bound monomer and SDS stable oligomers were then detected by
electrophoresis of the unheated samples in a 12% SDS-polyacrylamide gel
followed by autoradiography of the dried gel. The revertants of H35N
are marked with an asterisk (
), signifying that
they still contain Asn-35 as well as the designated mutation. Key: lane 1, WT-
HL; lane 2, H35N; lane 3,
F84L
; lane 4, I107M
; lane 5,
D108G
(isolate no. 11.1); lane 6,
T109I
; lane 7, E111G
; lane 8,
H144R
; lane 9, P151L
; lane
10, F153L
; lane 11, T155A
; lane 12, T155S
; lane 13,
V169M
; lane 14, S217C
; lane
15, L219P
; lane 16,
S222
; lane 17, F224Y
; lane 18,
D227N
; lane 19, D227A
; lane
20, F228L
(A); lane 21, F228L
(B). M, [
C]methylated protein markers (Life
Technologies, Inc.): myosin heavy chain (M
200,000), phosphorylase b (97,400), bovine serum albumin
(68,000), ovalbumin (43,000), carbonic anhydrase (29,000),
-lactoglobulin (18,400), lysozyme (14,300).
,
oligomeric
HL;
, monomeric
HL.
Figure 2:
Hemolytic activity and limited proteolysis
of the oligomerized HL-H35N revertants. For each
HL
polypeptide, the window shows a hemolysis assay of the intact molecule,
as monitored for 1 h at 20 °C in an automated microplate reader.
The in vitro translated protein was diluted 40-fold in the
assay mix. Below each assay trace is an autoradiogram of a 12%
SDS-polyacrylamide gel showing the proteolytic pattern of the
S-labeled
HL polypeptide after assembly on rRBC for 1
h at 20 °C at the same dilution used in the hemolysis assay.
Treatments were with water(-) or proteinase K (+) at 50
µg/ml for 5 min. Oligomers were dissociated by heating before
SDS-polyacrylamide gel electrophoresis. The revertants are designated
as described in Fig. 1(legend). The last two shown are, in
order, F228
L(A) and F228
L(B).
, undigested
HL; p, proteinase K
fragments generated by cleavage near the N
terminus(2, 3) .
Point mutagenesis, especially systematic scanning mutagenesis (20) , is valuable for obtaining information about the
functional roles of individual residues and short sequences of residues
in a polypeptide, provided that supporting evidence confirming the
structural integrity of the mutant molecules is obtained. A summary of
the results obtained by scanning point mutagenesis of HL is given
in the accompanying paper(4) , in which residues involved in
binding to rRBC, oligomerization, and pore formation are identified.
Despite its utility, point mutagenesis does not usually provide
definitive information about interactions between residues that lie far
apart in the linear sequence of the polypeptide chain. One way of
obtaining such information is to obtain second-site revertants of an
inactive protein by ``random''
mutagenesis(21, 22, 23, 24) . In
this study, we have located 16 residues that interact with the key
residue His-35 (3, 10, 11, 12, 13) by
seeking revertants of
HL-H35N, a mutant with greatly reduced lytic
activity. H35N is defective in the last step of hemolysin assembly:
conversion of a heptameric prepore to the fully active
pore(3, 13) . As hoped, we have found mutants in which
this step is repaired.
Nineteen independent revertants of H35N involving 16 amino acid residues were obtained by error-prone PCR. It must be noted that this method is far from ``definably random''(24) . First, only one base in a codon is changed and hence only a subset of the 19 possible amino acid substitutions can occur at each position. Second, Taq polymerase, as used here, is biased toward errors at AT base pairs; 17 out of 22 independent base changes occurred at AT pairs (Table 1). Further, transitions (15/22) were more frequent than transversions (7/22), which are twice as likely on purely statistical grounds.
In many cases, rather
subtle changes, e.g. Ser-217 Cys, Asp-227
Asn,
restored activity to
HL-H35N. This may reflect the fact that H35N
is poised at the brink of activity. H35Q, which contains an additional
methylene group is weakly active, while H35CamC, which contains an
additional -SCH
- group, has substantial
activity(13) . H35N is correctly folded as demonstrated by
limited proteolysis in solution, (
)and the defect is in a
single late step of assembly(3) . It seems likely that
reversion mutagenesis would be favored by such a situation of minimal
disablement. We were also aided by the development of a powerful
screening procedure and a high reversion frequency provided by the
large number of acceptable reversion sites.
Fourteen of the 16 amino
acids affected in the revertants are clustered in three regions of the
polypeptide chain (Fig. 3). Four revertants had mutations
between amino acids 107 and 111 inclusive, while five revertants (at
four amino acid positions) had mutations between amino acids 144 and
155. These two clusters flank the central loop, which plays an
important role in channel
formation(3, 4, 5, 8, 9, 25, 26) .
Further, Lys-110 and Asp-152, which are critical for lytic activity (4) , are located in the two clusters (Fig. 3). These
findings are in keeping with the demonstration that the N terminus of
HL and the central loop cooperate in the final step of
assembly(3) . Of five residues identified as crucial for pore
formation by cysteine scanning mutagenesis(4) , three are
interconnected by this study (His-35, Lys-110, and Asp-152). The
integrity of these three residues and Asp-24, which was not identified
here, is required for the final step of assembly. The fifth mutant,
E70C, is defective in binding and therefore would not be expected to be
linked with His-35. A third cluster of 8 revertants (at six amino acid
positions) is located between amino acids 217 and 228, a region that
was not explored in previous studies.
Figure 3:
Clustering of the mutations in the
HL-H35N revertants to three regions of the
HL polypeptide
chain. The polypeptide chain is shown as a linear sequence with the
glycine-rich central loop (residues 119-143) shaded. The five of
the 83 positions tested that yielded an inactive hemolysin when
substituted with cysteine (4) are indicated as large
circles: filled, forms heptamer but no transmembrane
channel (positions 24, 35, 110, and 152); unfilled, defective
in binding to rRBC (position 70). Position 35 is indicated as a large unfilled star and the sites of reversion as small
filled stars.
If the interactions revealed
by reversion mutagenesis take place within a single polypeptide chain,
the findings imply that the N- and C-terminal thirds of HL cannot
be considered as completely independent domains, although they contain
distinctive distributions of functional residues(4) .
Interactions between the N and C termini of monomeric
HL have been
demonstrated directly in experiments in which they are synthesized
separately and recombined to form a functional
hemolysin(25, 27) . Perhaps the regions around His-35
and residues 217-228 form a point of contact between the two
halves. Helix contacts in membrane proteins such as the a subunit of E. coli F
F
-ATPase (28) and the E. coli lactose permease (29, 30) have been proposed, based on the existence of
second-site revertants. Alternatively, because the fully assembled pore
is a heptamer, it is quite possible that intersubunit interactions are
corrected in the revertants.
Therefore, His-35 and all three
clusters may be in close proximity in the prepore (Structure 3), either
within individual subunits or at intersubunit contact sites, or they
may be brought into proximity during formation of the active pore
(Structure 4). Accordingly, the cysteine in H35C becomes unreactive
toward a water-soluble sulfhydryl reagent during formation of the
prepore(12) . Proximity of the residues in question would lend
a ready explanation for the revertants as beneficiaries of compensating
mutations that through direct interaction repair a defect in the final
step of assembly. However, it is by no means certain that the
restoration of activity is the outcome of such proximity. For example,
while second-site revertants of a defective triose phosphate isomerase
were clustered near the primary mutation at the active site (24) , reversion of other mutant proteins such as
staphylococcal nuclease (23) and phage lambda repressor (22) can be brought about by amino acid substitutions distant
from the primary site. Therefore, it will be most interesting to
examine the placement of His-35, the three clusters and the two lone
mutations at amino acid positions 84 and 169 in a three-dimensional
structure of HL.