PhD: University of Michigan
Postdoctoral training: Caltech and University of Illinois at Urbana-Champaign
Inhibitory Serpin Folding & Function
Inhibitory serpins regulate serine and cysteine proteases involved in inflammation, blood clotting, blood clot clearance (fibrinolysis) and other important physiological processes. The unique serpin inhibitory mechanism requires formation of a covalent bond between the protease and serpin, translocation of the protease relative to the serpin, remodeling of the serpin structure and deformation of the protease structure (see the figure above). In essence, inhibitory serpins behave as molecular mousetraps storing energy in their metastable native states and using this energy to deform and thus inhibit target proteases using a suicide mechanism. We are using single molecule fluorescence and other biophysical techniques to investigate both serpin-protease interactions and how serpins, particularly α1-antitrypsin (α1AT also known as α1-proteinase inhibitor) and antithrombin III (ATIII), fold to a kinetically trapped metastable state that is not the free energy minimum.
In cells, proteins can fold vectorially (from the N to the C terminus) as they are being synthesized, and folding may be assisted by molecular chaperones. Protein folding in vivo is also complicated by the myriad of other macromolecules (>100 mg/ml) present in cells, resulting in many possible non-specific interactions and reducing the space available to the folding protein. Thus, protein folding in the cell may be quite different than folding in the dilute solutions encountered in test tubes. In collaboration with Professors Lila Gierasch and Daniel N. Hebert at the University of Massachusetts Amherst, we have extended our inhibitory serpin folding studies into cells. Test-tube folding results from a variety of groups show that the proper packing of the serpin C-terminal region is critical for folding. ATIII in-cell folding studies confirm the importance of C-terminus packing and reveal that the C-terminus of ATIII is fully packed before the N-terminus. These results suggest that molecular chaperones could help choreograph serpin folding by delaying packing of the N-terminus.
Atomistic data on serpin conformational changes and serpin folding may be provided by molecular dynamics (MD) simulations, but this is difficult due to the large size (about 400 amino acids) of serpins. In collaboration with Pietro Faccioli (U. Trento, Italy) and Patrick L. Wintrode (U. Maryland School of Pharmacy), we have applied novel MD approaches developed by the Faccioli lab to simulate the active to latent transition of the serpin plasminogen activator inhibitor 1 (PAI-1) revealing the conformation of the partially loop inserted pre-latent intermediate. We are now using these computational methods to simulate serpin folding and to gain an atomistic understanding of how serpins fold and how disease associated mutations lead to misfolding.
How Do Peripheral Membrane Proteins Recognize & Bind to Membranes?
Peripheral membrane proteins (also called amphitropic proteins) interact with cell membranes modulating physical properties such as membrane curvature and influencing important physiological processes including signaling, immunity and infection. Using fluorescence correlations spectroscopy (FCS) and single molecule fluorescence microscopy we are investigating how the phospholipase C family of peripheral membrane enzymes recognize and bind to membranes. We are particularly interested in how phosphatidylinositol specific phospholipase C (PI-PLC) enzymes secreted by Gram positive bacteria such as Bacillus species and Staphylococcus aureus target mammalian cell membranes and how these virulence factors assist in infection. Our single molecule fluorescence and FCS studies of Bacillus PI-PLC interactions with lipid vesicles suggest that this PI-PLC preferentially binds to lipid packing defects which are more common in highly curved membranes. In collaboration with Mary Roberts (Boston College) and Nathalie Reuter (U. Bergen, Norway), we have demonstrated that despite sequence similarities Bacillus PI-PLC and S. aureus PI-PLC use very different mechanisms to recognize and bind to the outer membrane of mammalian cells. Phosphatidylcholine (PC) is an abundant zwitterionic phospholipid in the outer membrane of mammalian cells, and Bacillus PI-PLC specifically binds PC while S. aureus PI-PLC does not. Bacillus PI-PLC specifically recognizes and binds to PC using a cation-p cage where two or more aromatic residues, usually Tyr, interact with the cationic choline. This under-recognized motif is also used by other peripheral membrane proteins, and likely integral membrane proteins, to specifically bind PC.
Peripheral membrane enzymes must search two-dimensional membranes to find multiple substrates. Our single molecule fluorescence studies of Bacillus PI-PLC interactions with tethered lipid vesicles suggest that this PI-PLC uses a combination of short (100 msec) two-dimensional scoots on membranes combined with dissociation and three-dimensional excursions to search for substrates. This transient binding search strategy is consistent with our findings from FCS experiments where increased binding affinity can decrease enzyme activity. We are investigating whether other PI-PLCs use similar transient binding strategies to efficiently search for substrates on two-dimensional surfaces.
(Complete publication list at NCBI)
Chandrasekhar, K., Ke, H., Wang, N., Goodwin, T., Gierasch, L.M., Gershenson, A. & Hebert, D.N. (2016) Cellular folding of a metastable serpin. Proc Natl Acad Sci USA 113: 6484-6489. [PubMed]
Yang, B., Pu, M., Khan, H.M., Friedman, L.J., Reuter, N., Roberts, M.F. & Gershenson, A. (2015) Quantifying transient interactions between Bacillus phosphatidylinositol-specific phospholipase-C and phosphatidylcholine-rich vesicles. J Am Chem Soc 137: 14-17. [PubMed]
Cazzolli, G., Wang, F., a Beccara, S., Gershenson, A., Faccioli, P. & Wintrode, P.L. (2014) Serpin latency at atomic resolution. Proc Natl Acad Sci USA 111: 15414-15419. [PubMed]
Liu, L., Werner, M. & Gershenson, A. (2014) Collapse of a long axis: single molecule FRET and serpin equilibrium unfolding Biochemistry 53: 2903-2914. [PubMed]
Gershenson, A., Gierasch, L.M., Pastore, A. & Radford, S.E. (2014) Energy landscapes of functional proteins are inherently risky. Nat Chem Biol 10: 884-891. [PubMed]
Grauffel, C., Yang, B., He, T., Roberts, M.F., Gershenson, A. & Reuter, N. (2013) Cation-p interactions as lipid specific anchors for phosphatidylinositol-specific phospholipase-C. J Am Chem Soc 135: 5740-5745. [PubMed]
Cheng, J., Karri, S., Grauffel, C., Wang, F., Reuter, N., Roberts, M.F., Wintrode, P.L. & Gershenson, A. (2013) Does changing the predicted dynamics of a phospholipase C alter activity and membrane binding? Biophys J 104: 185-195. [PubMed]
Pu, M., Roberts, M.F. & Gershenson, A. (2009) Fluorescence correlation spectroscopy of phosphatidylinositol-specific phospholipase C monitors the interplay of substrate and activator lipid binding. Biochemistry 48: 6835-6845. [PubMed]
Farbman, M.E., Gershenson, A. & Licht, S. (2007) Single-molecule analysis of nucleotide-dependent substrate binding by the protein unfoldase ClpA. J Am Chem Soc 129: 12378-12379. [PubMed]
Lu, L., Mushero, N., Hedstrom, L. & Gershenson, A. (2006) Conformational distributions of protease-serpin complexes: a partially translocated complex. Biochemistry 45: 10865-10872. [PubMed]