Julia Bourg November 3 rd , 2011

1. Julia Bourg November 3rd, 2011

2. Extracellular Matrix and Fibronectin Fibronectin S-S F1 F2 F3 NTD

3. Molecular Organization of SfBI from S. Pyogenes A B PyOn5= 1F1 PyTw5= 2F1 PyTh5= 3F1 PyFo5= 4F1 PyFi5 = 5F1 S – Signal Peptide U – Non-homologous Region PRR – Proline Rich Repeats UR – Upstream Fn-Binding Region FnBR – Fn Binding Region W & M – Membrane Spanning Region PyOT5 =1F12F1 PyTT5 = 2F13F PyFF5 = 4F15F1

4. Previous study of S. aureusGives Insight on S. Pyogenes • FnBPs contain IDRs that interact 1-5F1 • Analogous study with S. aureus • FnBRs bind to NTD in a tandem β-zipper • S. pyogenesSfBI invades via a bridge with integrins • SfBI has 5 FnBRs that bind to NTD • SfBI-5 (truncation) CTD is 1F1 and can bind to all five F1 modules 1-5F1 S-S F1 F2 F3 NTD

5. NMR and SSP Results A. Chemical shifts for 1HN, 15N, 13C’, 13Cα , 13Cβ demonstrate SfBI-5 is an IDR – no stable secondary structure B. CTD demonstrates a relative propensity for β-strand formation.Analogous study with StreptoccusdysgalactiaeFnBR B3T agrees with this prediction of CTF and 1F1 recognition

6. Secondary Structure Propensity (SSP) • Complete resonance assignments for 1HN, 15N, 13C’, 13Cα , 13Cβ • Secondary chemical shift: - SSP score fore each Amino Acid, i, given by: ΔδXjobs= observed secondary chemical shift ΔδXjα/β = Average secondary chemical shift of fully formed secondary structure α/ β σjα/β = standard deviation of ΔδXjα/β • The summation over X is for all chemical shifts used and can include 13Cα, 13Cβ, 13C’, 1Hα, 1HN, and 15N. • In general 13Cα, 13Cβ, 13C’, 1Hα are useful for IDPs. -SSP Score of +1 or -1 indicates α-helix or β-strand respectively - A 0.5 score indicates a 50% conformer of disordered/ordered state

7. Conserved FnBR residues / HHM Model

8. ITC Results 4F1 4F1 WT K556E K556A WT 3.5± 0.2 nM3.5 ± 0.2 nM 3.5 ± 0.2 nM mSfBI-5 - - - - 0.8± 0.2 nM 1.6 ± 0.2 nM

9. ITC Results 3F14F1 4F1 M562A E553A T558A WT 3.5 ± 0.2 nM3.5 ± 0.2 nM 3.5 ± 0.2 nM mSfBI-5 8.2 ± 0.4 nM 8.4 ± 0.8 nM 55.4 ± 2.7 nM

10. Thermodynamic Parameters • Conserved FnBRresidue demonstrate weaker binding after the Alanine. With the exception of K556, where tighter binding was demonstrated. • Binding is across an interface and not hotspots (Phe -554, Thre-558, Gly-564, and Asp-574)

11. SPR Results ITC: Van’t Hoff Isochore to adjust the temperature difference 55.4 ± 2.7nM to 0.18nM

12. Crystal Structures PyTT5 and 2F1 and 3F1 C A, B. PyTT5 binds 2F1 and 3F1 through a tandem-zipper mechanism, with PyTT5 residues 562–565 and 570–574 adopting φ and ψangles typical of the β-strand. C. Comparison with 2F13F3·STATTI shows a σrms of 2.11Å

13. Proface Analysis of Interface of 2F13F1-PyTT5

14. Graphical Representation of Proface Analysis A. Interfaces of PyTT5 binding to 3F1 and 2F1 at the atom level. Buried forms (Red) and accessible forms (blue). B. Interface at the residue level, core (magenta) and rim (cyan) residues.

15. Model of the 1-5F1-SfBI-5 Complex SfBI-5 green, 1-5F1 purple

16. Conclusions • Demonstrated SfBI-5 is an IDR. CTD has a propensity to form β-strands when bound to 1F1 • Crystal structure PyTT5-2F13F1 complex demonstrates a tandem β-zipper. • Previous studies of SfBI with FnBRs also show the same motif on 4F15F1 and 1F12F1. Very likely SfBI-5 will do the same with 1F1 and 4F15F1. • Binding occurs over a large interface, not core hotspot residues. Confirmed with a HotPOINT analysis consistent with ITC results • Why are the residues conserved if not Hot Spots? May be attributed to maintaining the disordered state of FnBRs. Also limits the favorable interactions with their neighbors. • Distributing of binding over the interface adds more difficulty for the development of inhibitors.

17. Additional Figures

18. Hidden Markov Model (HMM) Probability of CGGSV = 0.8 * 0.4 * 0.8 * 0.6 * 0.2 = .031

19. Hidden Markov Model (HMM) ACCY Probability = .4 * .3 * .46 * .6 * .97 * .5 * .015 * .73 *.01 * 1 = 1.76x10-6