Coiled-coil motifs have been increasingly identified within a wide range of proteins and are believed to perform a variety of roles delivered by extended, relatively rigid structures (reviewed in Lupas, 1996
). Although many coiled-coil proteins are dimeric, parallel trimeric coiled-coil structures have been reported for a number of viral and bacterial proteins, including the influenza hemagglutinin (Wilson et al, 1981
; Bullough et al, 1994
), murine leukaemia virus spike protein (Fass et al, 1996
), mannose-binding protein (Sheriff et al, 1994
) and in the stalk of the head group of the UspA1-related protein YadA (Nummelin et al, 2004
). In all of these cases, the trimeric structure acts as a stalk to project other bio-active domains away from the pathogen membrane. In none of these cases, however, does the coiled-coil region itself act as ligand, instead playing a supporting structural role. The partial structure of UspA1 reported in this study is different. First, at about 200 Å in length (double the extended hemagglutinin structure), UspA1(527–665)
represents the longest stretch of trimeric coiled-coil for which a detailed structure is available. Second, UspA1 is unusual in utilising an extended stretch of regular coiled-coil as a receptor-binding domain. This is the first definitive example of this function for a trimeric coiled-coil, but it is likely there are others. For example, the M-like fibrinogen (Fg)-binding protein from the equine-specific bacterium Streptococcus equi
has also previously been reported to contain an Fc-receptor-binding region within a multimeric coiled-coil stalk (Meehan et al, 2002
), although no structural details are available.
The UspA1 protein is present at high density on the bacterial cell surface, and electron microscopy studies (Hoiczyk et al (2000)
and Supplementary data
) indicate this leads to forest-like densities in which the head groups may limit access to the closely packed stalk (trunk) regions. This arrangement is consistent with the linear structure observed for the uncomplexed form of UspA1(527–665)
within the crystal lattice; maximisation of crystal contacts would be expected to favour this form. At the bacterial surface, close packing of adhesins might be encouraged by the hydrophobic nature of the ligand-binding site and may, through restricting access of large antibodies, confer some protection of this region from immune recognition. This arrangement, however, presents a problem in that the resulting physical locations of the respective ligand/receptor pair seem incongruous to permit sufficiently close approach of bacterial and human cell membranes to allow effective adherence via the CEACAM1-binding site. Electron microscopy studies indicate UspA1 extends approximately 600–700 Å from the bacterial cell surface (Hoiczyk et al, 2000
). This is consistent with the expected length of an extended coiled-coil segment (residues 333–742) of which UspA1(527–665)
represents approximately one-third (200 Å). In the fully extended intact ligand, the receptor-binding site would therefore be located at least 450 Å from the membrane-distal end of UspA1. In contrast, its receptor site on human CEACAM1—by analogy with its similar construction to CD4 for which an overall structure is known (Wu et al, 1997
)—can reach no further than about 120 Å from the epithelial cell surface. Even if the receptor is primarily located within the highly invaginated microvilli of epithelial cells (Hammarstrom, 1999
) the sheer bulk created by the length of the extended head group would seem to present a steric difficulty for placing the ligand and receptor in close proximity. Two explanations seem possible. The head group of UspA1 may insert into the epithelial cell membrane prior to attachment of CEACAM1, allowing the base of the stalk region to come into closer proximity to epithelial cell surface. Alternatively, as suggested by this study, the stalk of UspA1 is capable of changing conformation to allow closer approach of the two respective membranes ().
Deformability of the UspA1 stalk region is demonstrated by the solution scattering studies, where in the presence of its CEACAM1 receptor UspA1(527–665) displays a distinctive curvature (bending angle of approximately 30–60°). This implies that in its receptor complex the UspA1 stalk has increased flexibility. Although self-aggregation prevents us from accurately interpreting similar solution scattering data collected in the absence of CEACAM1, the change in the scattering curve that accompanies addition of the receptor () is consistent with an alteration in the molecular structure. Further, as the crystal structure shows UspA1(527–665) to be symmetrical there appears to be no inherent mechanism for it to adopt a discrete bend (an asymmetric structure) in the absence of other effectors. This is supported by the molecular dynamics simulations, which indicate that the extended structure exhibits a degree of flexibility, but remains rod-like overall. In contrast, the same simulations conducted in the presence of bound N-CEACAM1 reveal a ‘weak spot' within the trimeric coiled-coil in the vicinity of residue 610, equidistant between the CEACAM1-binding site and the stutter and coincident with the incorporation of internally bound ions ( and ). Binding of N-CEACAM1, therefore, seems to provide a mechanism to ‘break' the three-fold symmetry and hence encourage formation of the bowed structure. The asymmetry produced by the bending is likely to distort remaining binding sites generated by the inherent three-fold symmetry, accounting for the observation that a maximum of two N-CEACAM1 molecules readily associate with each UspA1 trimer. Binding of receptor may also disrupt interactions of this hydrophobic region with neighbouring copies of UspA1 (akin to disruption of aggregation in the SAXS studies). The observation of curvature of UspA1 when in complex with its receptor noted in the solution scattering studies is supported by (i) the molecular dynamics simulations; (ii) the electron microscopy studies of the Mx cell surface where significant perturbations of the adhesin ‘forest' are observed and (iii) is a logical inference from the AUC and ITC measurements of a maximal stoichiometry of two molecules of N-CEACAM1 can bind with high affinity to each trimer of UspA1. In combination, these data support the concept of increased flexibility and the ability to adopt a distinctly bowed structure when UspA1(527–665) binds to its N-CEACAM1 receptor.
It is possible that this potential for the stalk region of UspA1 to bend within this discrete region may arise from the internal histidines and/or the stutter within the UspA1(527–665)
coiled-coil, which appear unique to this part of UspA1. The CD unfolding data highlight the marked instability of the coiled-coil structure to decreases in pH. Considering also the adjacent stutter, this whole region may be readily susceptible to the types of deformation seen in the SAXS analysis, molecular dynamics simulations and EM studies. By corollary, the extended triple coiled-coil in influenza hemagglutinin incorporates both buried histidines and several stutters along its length (Bullough et al, 1994
) and is believed to be metastable—allowing a dramatic re-modelling to take place as the pH is changed. The stimulus that instigates the conformational change in UspA1—whether chemical or mechanical—is unknown. It is possible that the composition of this region simply makes it more susceptible to deformation in response to mechanical force. Electron microscopy studies of UspA1 (Hoiczyk et al, 2000
and Supplementary data
) and the related protein EmaA (Ruiz et al, 2006
) at their respective cell surfaces indicate that a variety of conformations are observed but distinctive kinks are evident in both cases. Bending may be initially required to allow access for the receptor, which then stabilises this conformation by breaking the three-fold symmetry of the UspA1 stalk.
A recent structural determination of the E. coli
Dr adhesin bound to its human CEA receptor indicates that Dr also binds across the homo-dimer-forming surface of the receptor (Korotkova et al, 2008
). This is very similar to the surface of CEACAM1 eclipsed by UspA1 in the current study (Korotkova et al, 2008
). Note that the bacterium may achieve an advantage in targeting this largely invariant surface of the receptor. In both cases, it would be expected that adhesin binding would be accompanied by disruption of the homo- and heterophilic interactions known to occur between members of the CEACAM family (Watt et al, 2001
; Korotkova et al, 2008
UspA1 is also known to interact with laminin (Tan et al, 2006
), a component of the basement membrane, and with fibronectin (Tan et al, 2005
), which is prevalent in the extracellular matrix. Both these activities have been localised to the head group or nearby region. It is not known whether UspA1 can bind these ligands simultaneously in addition to CEACAM1. Further studies are required to provide a composite model for the multi-functional behaviour of UspA1, including its dynamic responses to the mechanical forces encountered as the bacterium encounters its host cell. Finally, UspA1(527–665)
has been shown to block binding of Mx
clinical isolates and to generate a protective immune response against all Mx
isolates tested (Hill et al, 2005
). From the identification of the N-CEACAM1-binding site and its molecular structure provided by these studies, it may now be possible to engineer smaller constructs with valuable therapeutic applications.