(Received for publication, July 13, 1995)
From the
Bacterial adherence to host tissue involves specific microbial
surface adhesins of which a subfamily termed microbial surface
components recognizing adhesive matrix molecules (MSCRAMMs)
specifically recognize extracellular matrix components. We now report
on the biophysical characterization of recombinant fibronectin binding
MSCRAMMs originating from several different species of Gram-positive
bacteria. The far-UV CD spectra (190-250 nm) of recombinant forms
of the ligand binding domain of the MSCRAMMs, in a phosphate-buffered
saline solution at neutral pH, were characteristic of a protein
containing little or no regular secondary structure. The intrinsic
viscosity of this domain was found to be the same in the presence or
absence of 6 M guanidine hydrochloride, indicating that the
native and denatured conformations are indistinguishable. On addition
of fibronectin NH terminus as ligand to the recombinant
adhesin there is a large change in the resulting far-UV CD difference
spectra. At a 4.9 M excess of the NH
terminus the
difference spectra shifted to what was predominately a
-sheet
conformation, as judged by comparison with model far-UV CD spectra. The
fibronectin NH
-terminal domain undergoes a minute but
reproducible blue-shift of its intrinsic tryptophan fluorescence on
addition of rFNBD-A, which contains no tryptophan residues. Since this
result indicates that there is no large change in the environment of
the tryptophan residues of the NH
terminus on binding, the
large shift in secondary structure observed by CD analysis is
attributed to induction of a predominately
-sheet secondary
structure in the adhesin on binding to fibronectin NH
terminus.
Many pathogenic bacteria have been shown to specifically
recognize and bind to various components of the extracellular matrix in
an interaction which appears to represent a host tissue colonization
mechanism. This adherence involves a group of bacterial proteins termed
MSCRAMMs ()(microbial surface components recognizing
adhesive matrix molecules)(1, 2) . A number of
Gram-positive bacteria have been shown to express fibronectin (Fn)
binding MSCRAMMs, and in some cases these proteins have been isolated
and the corresponding genes cloned and characterized. The primary Fn
binding sites in these MSCRAMMs have been localized to domains present
in most Fn binding adhesins. This domain is composed of a unit of
37-48 amino acids, repeated three or four times (Fig. 1A).
Figure 1: A, domain organization of fibronectin receptors from Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus pyogenes. Fn-binding repeats are represented by A, B, D, and P. S, signal sequences; U, sequence unique to the Fn receptor; W, cell wall spanning region; M, membrane-spanning region; C, intracellular sequence. The recombinant proteins correspond to the regions indicated by rFNBD-D, rFNBD-A, PAQ8, rFNBD-B, and rFNBD-P. B, aligned sequences of the Fn-binding repeat units indicated by A, B, D, and P in A. Regions of very high similarity are shown in bold type.
The repeat regions have been
overexpressed as recombinant fusion proteins in Escherichia coli where the recombinant Fn binding domains (rFNBD) are linked to a
stretch of histidine residues which are utilized for affinity
purification of the rFNBD proteins. These proteins have been designated
as rFNBD-D, rFNBD-A, rFNBD-B, and rFNBD-P, respectively (Fig. 1A). The rFNBDs were found to exhibit similar
binding kinetics and dissociation constants; for example, the
dissociation constants of the four recombinant proteins binding to
porcine Fn was determined by biosensor analysis to be in the low nM range with the dominant dissociation rates varying between 1
10
and 6
10
s
. Additionally, the recombinant proteins have
been shown to have cross-species specificity and inhibit binding of Fn
to many different bacterial cells(3) .
The repeated units of the fibronectin binding domains of the different MSCRAMMs are strikingly similar (Fig. 1B) and appear to contain a consensus sequence(4) . The repeat units have a high number of acidic residues, and there are conserved hydrophobic and acidic residues at certain positions. Overall there is a high degree of sequence similarity between repeated units in a specific MSCRAMM as well as between MSCRAMMs from different species. Our laboratory has determined that synthetic peptides, analogous to sequences shown in Fig. 1B, also bind Fn, and by amino acid substitution in these peptides it was determined that all conserved residues are not needed for Fn binding(4) .
Fn is a disulfide-linked dimeric
glycoprotein that is found in a soluble form in body fluids and a
fibrillar form in the extracellular matrix. The primary biological
function of Fn appears to be related to its ability to serve as a
substratum for the adhesion of animal cells. This adhesion is mediated
by a family of dimeric receptors which recognize and bind to specific
sites in the central part of Fn. The primary binding site in Fn for
MSCRAMMs from Gram-positive bacteria has been localized to the Fn
NH-terminal domain (N29)(5, 6) . This
domain is composed of five type I modules (FI) which are about 45 amino
acids in length. The structure of N29 is a series of anti-parallel
-sheets stabilized by several disulfide bonds interspersed at
regular intervals in the sequence(7, 8) .
Our laboratory is interested in the study of adhesin/host interactions with the ultimate goal to develop agents capable of blocking adherence of bacteria to the host as potential antibacterial therapeutics. The present study was initiated to gain insight into how the MSCRAMM interacts with fibronectin N29 at the molecular level. The conformational state of the recombinant ligand binding domain before and after N29 binding was explored by biophysical means.
where c is concentration and k is a dimensionless constant.
Figure 2: Far-UV CD of (from bottom to top at 200 nm) PAQ8, rFNBD-P, rFNBD-A, rFNBD-B, and rFNBD-D.
Both rFNBD-A and rFNBD-D were tested to determine if the proteins exhibited some degree of regular secondary structure elements that could be disrupted on thermal denaturation. For each protein sample the temperature was raised from 24 to 80 °C and far-UV CD spectra were accumulated at several different temperatures (data not shown). No conspicuous differences were seen in any of the spectra, indicating that there was not a significant amount of secondary structure present that was unfolded during heating.
Because of the apparent lack of stabilizing secondary structure
elements, the possibility that the rFNBD proteins had a highly
fluctuating tertiary structure in buffers approximating physiological
conditions was investigated. One method of observing the stability of
tertiary structure is to monitor the cooperativity of a protein
unfolding transition. The term cooperativity describes the process in
which weak intramolecular interactions cooperate so that the
interacting groups have very high effective concentrations, thus
stabilizing a particular folded conformation. As intramolecular
interactions are disrupted the protein unfolds completely within a
limited range of condition changes, and the abruptness of the unfolding
is indicative of a cooperative transition(17) . By monitoring
the elution volume (V) of rFNBD-B using
gel-permeation chromatography as the concentration of GdnHCl was
increased from 0 to 6 M, it was determined that rFNBD-B does
not follow a cooperative unfolding model (Fig. 3). The
cooperative unfolding transition of a globular protein of comparable
molecular mass, ribonuclease A (13.7 kDa), is shown in Fig. 3for comparison. It is readily apparent that ribonuclease A
undergoes a distinct shift in V
during its
unfolding transition between 2 and 4 M GdnHCl as previously
shown by Greene and Pace (13) .
Figure 3: Denaturation of rFNBD-B in increasing concentrations of GdnHCl (open circles). The denaturation curve of ribonuclease A (closed circles)(13) , a globular protein of comparable molecular mass, was also monitored for comparison.
The V (9.6 ml) of rFNBD-B at 0 M GdnHCl corresponds to a
molecular mass of
57,000 Da, when compared to a curve developed
using globular protein standards (ribonuclease A, chymotrypsinogen,
ovalbumin, and albumin). Because of the abnormally low V
, there is the possibility that rFNBD-B is
aggregated under these conditions, may not possess a compact globular
structure, or potentially some combination of the two possibilities.
When compared to a curve developed from the same set of four standard
proteins denatured in 7 M GdnHCl, the experimentally
determined molecular mass of rFNBD-B (13.3 kDa) in the absence of
GdnHCl corresponded reasonably well to the actual molecular mass of
rFNBD-B (14.6 kDa) (data not shown). This supports that rFNBD-B is an
unfolded protein in PBS.
To further ensure that aggregation was not
the cause of the abnormally low V of rFNBD-B in
the gel-permeation chromatography experiment, the intrinsic viscosity
of rFNBD-B and rFNBD-A was determined in the absence and presence of 6 M GdnHCl (Fig. 4). The intrinsic viscosity
([
]) is a measure of the effective specific volume of
the domain of a macromolecule in solution(14) . For folded
globular proteins, [
] is small and independent of
molecular mass, while [
] for denatured proteins is
considerably larger and increases roughly as the molecular mass of the
monomer unit increases. The specific viscosity (
) of
rFNBD-A and rFNBD-B at concentrations of 0 and 6 M GdnHCl was
measured. By plotting
/c versus c (), it was determined that the [
] was
the same for both rFNBD-A and rFNBD-B in the absence or presence of
GdnHCl (Fig. 4). This result demonstrates that both rFNBD-A and
rFNBD-B are monomers at 25 °C. Additionally, it is further evidence
that both rFNBD-A and rFNBD-B do not significantly change conformation
by changing from 0 to 6 M GdnHCl. That the proteins are in
fact monomeric in PBS at 18-20 °C has subsequently been
confirmed by both velocity and equilibrium sedimentation analysis using
a Beckman XL-A analytical ultracentrifuge. (
)
Figure 4: Viscosity data for (A) rFNBD-A in PBS (open circles) and 6 M GdnHCl (closed circles) and (B) rFNBD-B in PBS (open circles) and 6 M GdnHCl (closed circles).
One of the characteristics of partially folded proteins is the presence of pockets of hydrophobic side chains that are sequestered away from the highly polar solvent. Intrinsic tryptophan (Trp) fluorescence of rFNBD-B in PBS indicates that the single Trp residue is not in a hydrophobic region and on addition of 6 M GdnHCl there is no change in the wavelength or intensity of the maximum fluorescence for this Trp (data not shown). A convenient probe for monitoring the presence of hydrophobic pockets in partially folded proteins is ANS. ANS is a fluorophore that emits a large amount of fluorescence on binding to partially exposed hydrophobic regions. These regions are not normally present in tightly packed native proteins because their hydrophobic clusters are buried in the interior and are not accessible to solvent. Likewise, denatured proteins are known not to bind ANS(18) . It was determined that ANS did not bind to rFNBD-A or rFNBD-B under any conditions tested, indicating that the proteins do not appear to be capable of forming localized, stable regions of hydrophobic side chains (data not shown). From the experimental evidence presented above we conclude that the rFNBDs do not have the characteristics of folded or partially folded (i.e. a molten globule) proteins, instead the structure of the repeat regions is highly dynamic and appears to be thermodynamically indistinct from the denatured state.
Figure 5: Intrinsic tryptophan fluorescence of 3.5 µM N29 in the absence (solid line) and presence of a 2.5 excess molar ratio of rFNBD-A (dashed line).
The far-UV CD of the N29 fragment is shown
in Fig. 6A and it agrees well with previously published
spectra(22) . The N29 fragment exhibits an unusually low
intensity far-UV CD and the observed positive band at 230 nm has
been indicated to arise predominately from the optical activity of
tyrosine side chains in N29(23, 24) . A comparison of Fig. 2and 6B indicates that the maximum intensity of
the [
] values (190-250 nm) of N29 are
significantly smaller than those for the rFNBD proteins.
Figure 6: A, far-UV CD of N29 (10.5 µM). B, far-UV CD of the titration of 8.6 µM rFNBD-A with increasing concentrations of Fn N29. The N29 concentrations are (from bottom to top at 200 nm) 0, 10.5, 21, 32, and 42 µM.
Far-UV CD
has been used on a variety of systems to gain information about
secondary structure changes that take place in a protein on binding to
various biological molecules (see for example, (19, 20, 21) ). On addition of N29 to rFNBD-A
at ratios ranging from 0:1 to 4.9:1 there is a marked change in the
appearance of the far-UV CD spectra. The mean residue ellipticity
([]) of rFNBD-A at the 0:1 ratio is -23,500
deg
cm
/dmol at 200 nm. The Fn N29 is then added and a
new spectrum is acquired. After the N29 spectrum in isolation is
subtracted from the mixture spectrum, [
] is calculated
for the subtracted spectrum using the mean residue molar concentration
for rFNBD-A only (Fig. 6B). Treating the N29 spectrum
as a constant and subtracting it from the mixture spectrum is justified
by the presence of ordered secondary structure in N29 (22, 25, 26) as well as the lack of evidence
for a significant conformation rearrangement in N29 on binding of
rFNBD-A, as judged by fluorescence spectroscopy.
At a ratio of 4.9:1
the resulting spectrum now has a maximum [] of
+17000 deg
cm
/dmol at 200 nm and a minimum
[
] of -6000 deg
cm
/dmol at 230
nm. The same changes in the far-UV CD spectra of rFNBD-D and PAQ8 was
seen when each was titrated with N29 (data not shown). The rFNBDs
appear to change structure from a predominately random coil
conformation in an unoccupied form to a predominately
-sheet
secondary structure conformation in the MSCRAMM ligand complex.
Several controls were examined to ensure the changes seen in Fig. 6B were due to a binding event and not nonspecific hydrophobic interactions, due to rFNBD-A existing in solution as an unfolded protein. The far-UV CD spectra of either rFNBD-A or N29 in the presence of bovine serum albumin were identical to those acquired in the absence of bovine serum albumin. Moreover, when a 2-fold molar excess of N29 was combined with a relatively unstructured recombinant segment of the collagen-binding MSCRAMM (28) there was no evidence of any type of interaction as measured via this assay.
The rFNBD proteins appear to have little or no stable
secondary structure in PBS. A distinction between native and denatured
tertiary conformations was not measurable using intrinsic viscosity
measurements. Tanford and co-workers (14) have shown that there
is a linear relationship between the intrinsic viscosity
[] of a denatured protein and the number of amino acids
in its polypeptide chain (n) as shown by :
From the [] of denatured rFNBD-A
and rFNBD-B is calculated to be 18.8 and 18.2 ml/g respectively. This
value is in agreement with the experimental [
] values
extrapolated from Fig. 4(16.1 and 17.1 ml/g for rFNBD-A, and
17.8 and 18.0 ml/g for rFNBD-B) under native and denaturing conditions,
respectively. This result would suggest that the rFNBD proteins have
the same highly dynamic structure in either solvent. Furthermore, these
proteins do not aggregate and are essentially monomeric at 25 °C in
PBS. The absence of fixed long-distance intramolecular interactions is
confirmed by the results of unfolding experiments monitored by
gel-permeation chromatography. For rFNBD-B in increasing concentrations
of GdnHCl any structural changes occur in a gradual, linear, fashion
without a clearly identifiable transition region, as seen for the
control, ribonuclease A.
The solution structure of two of the five
type I (FI) repeats present in the N29 portion of Fn has been
determined in isolation and as a pair using multidimensional nuclear
magnetic resonance spectroscopy(25, 26) . The repeats
are made up of a series of anti-parallel -sheets where a single FI
module consists of an amino terminus leading into a short
double-stranded sheet and folded onto a larger triple-stranded sheet to
enclose a hydrophobic core. Each FI module is further constrained by
two conserved cysteine residues that link in a pattern Cys
to Cys
and Cys
to Cys
.
Solution structure information indicates that the fourth FI module
docks onto the fifth FI module via a hydrophobic interface. The four
-sheets stack on top of each other to form a fairly inflexible
elongated rod-like structure that has only a limited clockwise twist
around the long axis from the NH
- to the COOH-terminal.
From electron microscopy of rotary shadowed specimens it was also
deduced that the entire Fn NH
-terminal domain is most
likely a rigid rod-like structure(22) . Because of the defined,
stable, secondary and tertiary structure in N29, the minute change in
the intrinsic Trp fluorescence in N29 on binding of rFNBD-A, and the
apparent lack of stable ordered conformation in the rFNBD proteins, the
large conformational shift seen in the far-UV CD on recombinant adhesin
binding to N29 is largely attributed to the rFNBD proteins assuming a
predominately
-sheet structure on binding to N29.
To what
extent might conformational changes in the N29 be contributing to the
changes on binding seen in Fig. 6B? Previously far-UV
CD has been used to monitor structural changes in a 31-kDa
NH-terminal tryptic fragment of Fn(N31)(21) . They
recorded reproducible changes in the secondary structure of N31 when
the polysaccharide heparin was added to the protein. On binding of
heparin, the signal at 228 nm in the far-UV CD of N29 (shown in Fig. 6A) is slightly red-shifted and attenuated.
Additionally, a minimum centered at 212 nm is accentuated. These
effects are of much less magnitude and opposite direction when compared
to Fig. 6B. We cannot discard the possibility of
changes in the far-UV CD of N29 on binding to the rFNBD proteins. Since
the secondary structure of the N29 repeat motifs is well ordered, the
most likely place for a significant conformational change to occur is
the segment of the polypeptide chain connecting the repeats. If there
is rearrangement in this region on binding of the adhesin we believe
that it would still not be sufficiently large to cause the shift from a
random-coil to a predominately
-sheet secondary structure seen in Fig. 6B.
Several pieces of evidence combine to suggest that the binding site between these proteins may be a series of multiple contact points along one or both of the repeat regions. The binding sites on both Fn and the adhesin consist of repeated sequences. Inhibition studies have shown that synthetic peptides analogous to some of the single repeat regions (Fig. 1B) retain the ability to bind Fn(11) . Additionally, repeats D1-3 expressed as a glutathione S-transferase fusion protein (D1-3) have recently been fluorescein-labeled and assayed for changes in the fluorescence anisotropy of this tag on addition of N29(27) . They determined that 1.9 mol of N29 bound per mol of D1-3 with a dissociation constant of 1.5 nM, indicating that there is at least two distinct binding sites on the D1-3 repeat region capable of binding N29. Finally, the overwhelming conformational shift shown in Fig. 6B also indicates that binding affects the entire recombinant adhesin domain and is not localized to a single area.
The conformational variability of the rFNBD proteins and PAQ8 as well as the observed change in conformation for the rFNBD proteins on binding of N29 does not appear to be the result of expressing this portion of the adhesin as a recombinant protein. A monoclonal antibody, 3A10, has been raised against the full-length MSCRAMM FnbA and characterized(9) . The epitope for this antibody has been mapped to the Au region of FnbA, which has also been shown to be most similar to the A2 sequence of FnbA and the P repeat motif, from S. pyogenes, and to bind Fn. However, 3A10 will only recognize and bind to the epitope in both full-length and truncated MSCRAMM protein when both components are in the presence of Fn. Additionally, monoclonal antibody 3A10 significantly enhances the binding of a corresponding epitope, present on the surface of S. pyogenes cells, to Fn(9) . Combined, these observations indicate that structural changes reported in this paper could mimic the molecular basis for the emergence of the epitope recognized by 3A10 in the intact MSCRAMM. The microbe may have developed the capability to circumvent the effects of blocking antibodies produced by the host by presenting the biologically relevant conformation only when the MSCRAMM is already bound to its ligand of choice. The ligand induced binding site could then be of significant advantage for the microbe in avoiding host defense mechanisms because the host is unable to mount a response that includes blocking attachment of the organism to Fn and establishing colonization.