| dc.description.abstract | Pore forming toxins (PFTs) belong to a class of bacterial exotoxins that disrupt the
biological membrane barrier by formation of nanopores. These toxins are central to
the virulence of pathogens such as S. pneumoniae, V. cholerae, L. monocytogenes
and B. anthracis to name a few. Formation of pores lead to the killing of cells due
to cellular ion imbalance and efflux of important biomolecules such as ATP, from the
cell. Unique to this class of proteins is their ability to exist in bi-stable states i.e.,
they are produced in soluble forms and upon exposure to membrane, undergo conformational
changes, oligomerization and form a stable membrane integrated structure.
This spontaneous transformation occurs without the aid of any extrinsic processes
such as post-translation modifi cation, tethering to membrane anchors and translocon
mediated membrane insertion.
Self-assembly and bi-stability being intrinsic properties of PFTs, are embedded in
sequence and structure. However, barring a few PFTs, molecular details of regions
in the protein that are essential for the aforementioned processes have not been well
characterized. These questions are explored in an -PFT, Cytolysin A (ClyA) from
E. coli. ClyA is produced as a monomer and upon membrane exposure, undergoes
conformational changes and nally oligomerizes to form a dodecameric pore. It is
one of the few PFTs for which X-ray crystal structures are available, for both the
soluble and membrane inserted pore form. This information is valuable to correlate
structure, sequence, PFT self-assembly pathway and lytic action.
In the rst part of the work, the role of solvent-exposed C-terminal loop, comprising
of 11 amino acids, is examined. This region is devoid of any intra-protein contacts
in the monomer and neither is it speculated to have any inter-protein contacts in the
pore structure, however its deletion renders the protein inactive. Absence of any interactions
in the initial and final states implies that it is essential in the intermediary
steps of pore assembly. To identify the mechanistic details of modulation of activity,
the different steps of pore formation were monitored in a mutant devoid of the
C-terminal loop. Studies on binding and detergent-induced oligomerization did not
indicate any anomaly in comparison to the wild type protein. Nevertheless mutant
protein was found to be inactive. Thermal unfolding experiments revealed that deletion
of the C-terminal loop destabilized the structure of the monomer. Furthermore,
by using novel methods to reconstitute detergent-induced oligomers into vesicles, and
examining oligomer stability, it became clear that the instability in the monomer
was propagated to the oligomers as well. Hemolytic activity of the wild type protein
reduced signi ficantly when co-incubated with the C-terminal deletion mutant, possibly
due to sequestration of wild type units into inactive hetero-oligomers with the
mutant protein. Thus, instability in the monomer due to deletion of the C-terminal
loop, altered the delity of the conformational transition upon membrane exposure.
This resulted in formation of aberrant oligomers on the membrane surface. Therefore,
regions of the PFTs, not directly involved in the pore structure, appear to assist
in transitioning through intermediary steps of assembly, leading to successful pore
formation in a membrane environment.
PFT assembly on membrane is dependent on lateral motion of the toxin monomers,
as they directly impinge on collision frequency and hence oligomerization. However,
all studies to examine ClyA assembly have been carried out in solution, with detergent,
wherein micellar aggregation is the dominant driving force rather than toxin
self-assembly. Toxin self-assembly on the other hand is contingent on establishing
inter-protomer contacts and is influenced by lateral motion of the individual protein
molecules on the surface. However, little is known about PFT mobility on the
membrane surface and its implication in the formation of higher-order structures. In
the third Chapter, the lateral motion of ClyA on membrane was analyzed by using
single-molecule
fluorescence and spectroscopy. Single-particle tracking of ClyA was
carried out on polymer-cushioned, arti ficial membrane systems. Diffusional analysis
of particle trajectories revealed the existence of two discrete mobility states exhibiting
fast and slow dynamics. In the presence of membrane cholesterol, the population
of the slower moving species increased, with the concomitant decrease in the
fast mobility population. Analysis of transition probabilities between the mobility
states revealed that the conversion from fast to slower mobility state was due to the
conformational transition from a peripherally-associated protein conformation to a
membrane-inserted conformation respectively. Furthermore, a cholesterol consensus
and recognition (CRAC) motif was discovered in the transmembrane helix of ClyA.
Upon disruption of this motif, the fraction of fast moving species increased signi -
ficantly and activity of the mutant protein was signi ficantly compromised. This validated
that the conformational transition to form the membrane bound structure was
stabilized by the interaction with cholesterol. The presence of membrane cholesterol
triggered the insertion of the transmembrane helices leading to the formation of a
functional pore, thus providing a strategy for selective pore formation, in eukaryotic
membranes. Additionally, it serves as evidence that heterogeneous motion on the
membrane need not necessarily arise from the presence of `lipid-microdomains', and
conformational plasticity is also an important determinant of protein lateral motion.
In summary, this study has provided novel molecular insights into hitherto unknown
regulatory mechanisms of ClyA pore formation such as the C-terminal loop and
CRAC motif. Albeit seemingly disparate, the solvent exposed and transmembrane
domains appear to regulate different aspects of the assembly pathway for successful
pore formation. The results presented in the thesis allow reconciliation of biologically
relevant mechanism of virulence factor dissemination and activation on target cells.
In addition to enhancing the understanding of mechanisms in
uencing toxin function,
results presented here can enable design of novel `anti-toxin' therapies which can used
in case of pathogenesis. | en_US |
