1. Cystic FibrosisTransmembrane Conductance Regulator and Filamin A Background for “Biochemical Basis of the Interaction between Cystic Fibrosis Transmembrane Conductance Regulator and Immunoglobulin-like Repeats of Filamin” Smith et al. JBC 285 (2010): 17166-17176. Web. Presented by Amanda Maez March 9, 2011

2. Cystic Fibrosis Most common, lethal genetic disorder in Caucasians characterized by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) of epithelial cells Characterized by high sweat chloride concentration and dehydrated viscous secretions Most common mutation is the  F508 ~30,000 individuals in North America are affected (70% carry one copy of  F508)

3. Presentation Overview  CFTR Location ABC transporter- general structure Structure and Function Mutations  Filamin A  Actin binding protein (ABP)  Structure  Basis for Research of Smith et al.  Mutants are rapidly degraded in ER  S13F mutation disrupts CFTR-filamin interaction

4. CFTR is an ABC Transporter ATP binding cassette transporter 2 Membrane spanning domains (MSD1 and MSD2) 2 Nucleotide binding domains (NBD1 and NBD2) Motor Humans have at least 48 (3-5% of bacterial genome) K P Locher et al. Science 2002;296:1091-1098

5. CFTR Structure 2 MSDs 6 membrane-spanning  -helices in each 2 NBDs Each possess an ATP binding pocket ABP1 formed by Walker A and B motifs of NBD1, ABP2 by Walker A and B motifs of NBD2 Unique regulatory (R) region Located between the NH 2 terminal NBD and the second MSD

6. Structure of CFTR Chen, Tsung-Yu, and Tzyh-chang Hwang. "CLC-O and CFTR: Chloride Channels Evolved From Transporters." Physiological Reviews 88 (2008): 351-87. Web

7. CFTR Function Conducts Cl¯ across membrane when both NBDs have bound ATP and R domain is phosphorylated by protein kinase A Closes when ATP is hydrolyzed on one of the NBDs and R domain is no longer phosphorylated. Lehninger. Principles of Biochemistry. 5th Edition. W.H. Freeman and Company, 2008. 401. Print.

8. Mutations in CFTR Most common is the  F508, which is located in NBD1 Cause misfolding of the protein which lead to a defective channel due to inability to hydrolyze ATP Decrease in Cl¯ export is accompanied with a decrease in export of water and leads to thick, sticky mucus which is a haven for bacteria that are the ultimate cause of mortality These mutated CFTRs are rapidly transported to and degraded in the ER Those that are not degraded are usually subject to inefficient trafficking to the apical plasma membrane

9. Filamin A Structure High molecular weight Long rod-like domain of 24 repeated anti- parallel  -sheets (resembling immunoglobulin domain) Two flexible loops (30 aa) that form hinge structures

10. Fln A Structure Crystal Structure of C2 Fragment of Steptococcal protein G in complex with FC domain of Human IgG  Details of FlnA-Ig21:CFTR 4-22 crystal structure Sauer-Eriksson et al. Structure 3 (1995). Web. Smith et al. JBC 285 (2010), Supplemental Figures.

11. Filamin A Function Actin Binding Protein (ABP) F-actin crosslinker--scaffolding protein Anchors a variety of transmembrane proteins to the actin cytoskeleton

12. Fln A Function Nakamura et al. JCB 179 (2007): 1011-1025. Web.

13. Basis for Research of Smith et al. Mutated CFTRs are rapidly transported to and degraded in the ER Those that are not degraded maintain partial function, but are usually subject to inefficient trafficking to the apical plasma membrane Filamin A anchors CFTR to the actin skeleton Understand the interaction between Filamin A and CFTR using a mutation (S13F) that disrupts this binding

14. Sources Chen, Tsung-Yu, and Tzyh-chang Hwang. "CLC-O and CFTR: Chloride Channels Evolved From Transporters." Physiological Reviews 88 (2008): 351-87. Web. Feng, Yuanyi, and Christopher Walsh. "The Many Faces of Filamin: A Versatile Molecular Scaffold for Cell Motility and Cignaling." Nature Cell Biology 6.11 (2004): 1034-038. Web. Nakamura et al. “Structural Basis of Filamin A functions.”JCB 179 (2007): 1011- 1025. Web. Locher, Kaspar. "The E. Coli BtuCD Structure: a Framework for ABC Transporter Architecture and Mechanism." Ribbon diagram of the BtuCD protein structure. Science 296 (2002): 1091-098. Web Image. Sauer-Eriksson, A.E. "Crystal Structure of the C2 Fragment of Streptococcal Protein G in Complex with the Fc Domain of Human IgG." Structure 3 (1995): 265-78. RCSB Protein Data Bank. Web. 8 Mar. 2011. Smith et al. “Biochemical Basis of the Interaction between Cystic Fibrosis Transmembrane Conductance Regulator and Immunoglobulin-like Repeats of Filamin.” Journal of Biological Chemistry 285 (2010): 17166-17176. Web. Uribe, Ricardo, and David Jay. "A Review of Actin Binding Proteins: New Perspectives." Molecular Biology Reports 36 (2007): 121-25. Print.