SUPPLEMENTARY INFORMATION

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1 Supplementary Information to: Structural basis for the selectivity of the external thioesterase of the Surfactin-Synthetase Alexander Koglin 1,2, Frank Löhr 1, Frank Bernhard 1, Vladimir R. Rogov 1,3, Dominique P. Frueh 2, Eric R. Strieter 2, Mohammad R. Mofid 4, Peter Güntert 1,5, Gerhard Wagner 2, Christopher T. Walsh 2, Mohamed A. Marahiel 4 and Volker Dötsch* 1 1 Institute of Biophysical Chemistry, J.W. Goethe-University, Frankfurt am Main, Germany 2 Dept. of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA 3 Institutes of Protein Research Puschino, Russia 4 Dept. of Chemistry/ Biochemistry, Philipps University, Marburg, Germany 5 Frankfurt Institute for Advanced Studies, Frankfurt am Main, Germany * Corresponding author: vdoetsch@em.uni-frankfurt.de Based on the available information from NOESY spectra showing two sets of partially different NOEs and the specific selection of one state by complex formation with the native substrate (Supplementary Fig. 4), we built models of the assumed two individual states of the SrfTEII enzyme most likely representing an open and a closed state (Supplementary Fig. 7). The two models differ slightly in the angle of the C-terminal helix and noticeable in the helix-turn-helix motif. In the closed state, both elements show an inward movement leading to a more compact structure in which the active site is less accessible. In the open state the residues of the active site are less buried and become accessible to the solvent. This opening also uncovers an interaction surface (Fig. 3a), which allows a tight complex formation with the misprimed carrier protein (Fig. 3b). As discussed in the main text, these models are based on the assumption of only two different conformations and on guesses which stretches of double resonances belong to which conformation. 1

2 Supplementary Figure 1 Assigned 15 N HSQC spectra and conformational exchange of SrfTEII. The 15 N-HSQC spectrum was recorded on a Bruker Avance NMR-spectrometer operating at 900 MHz proton frequency equipped with a cryogenic 5 mm z-axis gradient triple resonance probe at 298 K. DSS (4, 4-dimethyl-4-silapentane-1- sulfonate) was used as internal chemical shift reference. The spectral width was set to 12.5 ppm in the proton and 35 ppm in the nitrogen dimension. A total of 2048 points in the direct and 1024 points in the indirect dimension were collected. Conformational exchange is evident from the existence of doubled signals for N-H cross peaks or considerable line broadening of single amino acid residues. In b the area of some of these double peaks is shown as enlarged inserts in the fully assigned 15 N-HSQC spectrum of SrfTEII. In a amino acid residues showing either double peaks (highlighted in red) or broad lines (colored gradient from yellow to orange) are shown on a ribbon presentation of the mean structure of SrfTEII. 2

3 Supplementary Figure 2 Structural bundle and secondary structure of SrfTEII. The CYANA 1,2 derived structural bundle of 20 models of SrfTEII is shown from its front view (a) and rotated by 180 (b). The structure is colored from N-terminus (blue) to C-terminus (red) and shows an α/β-hydrolase fold with a conserved seven stranded β-sheet packed between 4 structurally conserved α-helices, excluding the sequentially and structurally diverse helix-turn-helix or lid region (green). In c the secondary structure and solvent accessibility (white: accessible; blue: buried) are shown. The presentation of the mean secondary structure for the CYANA derived set of 20 structures of SrfTEII and the solvent accessibility is taken from PROCHECK ,4. 3

4 Supplementary Figure 3 Chemical shift deviations detected in titration experiments. The combined 15 N and 1 H chemical shift deviation of 15 N-HSQC based titration experiments 5 of 15 N-labeled SrfTEII with unlabeled Myristoyl-CoA (a), unlabeled holo-tycc3-t domain (b), unlabeled holo-tycc3-t domain loaded with a 4 -PP cofactor mimic, where the thiol is replaced by an amine to prevent hydrolysis by the thioesterase 6-8 and modified with Alanine as a substrate analogue (c) and holo-tycc3-t domain loaded with a non-hydrolysable 4 -PP cofactor mimic modified with the tripeptide PheProPhe (Chi Scientific, Maynard, USA) (d) are shown. The specific binding of Myristoyl-CoA by SrfTEII (a) supports the previously assumed second function of SrfTEII as the starter enzyme, being involved in the loading of the β-hydroxy-fatty acid onto the first C-domain 9. The recognition involves the regions around the active site residues of the catalytic triad (Ser86, Asp189 and His216) and the loop region around Gly140 and Gly141 within the sequentially diverse helix-turn-helix motif. No recognition by the 4

5 SrfTEII or rather no chemical shift changes for this titration experiment could be observed for the loop (Gly49 Thr59) surrounding the N-terminus. The interaction of SrfTEII with holo-tycc3-t domain (b) is comparably weak and involves the loop connecting Gly49 and Thr59. This loop region seems specifically involved in the recognition of T domains. The chemical shift deviation for Ser86 is set to the maximum and indicated as a red bar, since no chemical shift perturbation could be measured in the complex, due to severe line broadening. The comparison with the diagrams representing the NMR titration experiments with Ala-NH-holo-TycC3-T domain (c) and PheProPhe-NH-TycC3-T domain (d) reveals the selectivity for small substrates by the SrfTEII, as already demonstrated by kinetic data 10. The interaction of SrfTEII with PheProPhe-holo-TycC3-T-domain is limited to residues involved also in holo T-domain recognition, which supports the described selection of small substrates driven by the limited size of the cavity. For residues with resonances broadened beyond detection in the 1:1 SrfTEII, Ala-holo-TycC3-T-domain complex, the chemical shift deviation is set to the maximum and indicated as red bars. Chemical shift differences of amino acids showing chemical shift perturbations are shown as blue bars. 5

6 Supplementary Figure 4 Specific recognition of the native substrate by SrfTEII. The combined 15 N and 1 H chemical shift deviation of 15 N-HSQC based titration experiments of 15 N-labeled SrfTEII S86A with unlabeled acetyl-holo-tycc3-t domain is shown in the diagram. The comparably large chemical shift perturbations indicate a tight complex formation and specific recognition of the native substrate by SrfTEII. The comparison of the 15 N-HSQC spectra of the free (blue) and complexed (red) SrfTEII also demonstrates the selection of one of the two observed states in the 1:1-complex. The coloring of the surface (Δppm: > 0.2: blue; residues showing complete suppressed lines: red) indicates the compact recognition and interaction surface of the SrfTEII protein. Titrations of the mutant SrfTEII S86A enzyme with holo-t showed virtually the same results as the titration of wild type SrfTEII with holo-t, demonstrating that the stronger interaction with the acetyl-holo-t domain is not caused by the mutation. 6

7 Supplementary Figure 5 Structural comparison of SrfTEI and SrfTEII. The crystal structure of SrfTEI 11 (a; PDB code: 1JMK) compared to the NMR solution structure of SrfTEII (b) shows significant differences in the N-terminal part including the relative position of the first two β-strands. Both structures are color coded from blue (Nterminus) to red (C-terminus). The central figure represents the superposition of the full β-sheet and the helix-turn-helix motif of SrfTEI (shown in red) and the SrfTEII (shown in blue) proteins and highlights the repositioning of the shortened helix-turn-helix motif of the SrfTEII protein. 7

8 Supplementary Figure 6 Structural comparison of the active site cavities of SrfTEII and SrfTEI. The active site of the SrfTEII enzyme (a) is located in a shallow groove and is ideally suited to interact with small molecules attached to the 4 -PP cofactor of the holo-t domain. In contrast the crystal structure of SrfTEI 11 (b; PDB code: 1JMK) shows a deep and wide cavity that can accommodate the entire peptide attached to the cofactor. The catalytically active residues of both enzymes are colored in red. 8

9 Supplementary Figure 7 Structural Models of the opened (green) and closed (red) state of SrfTEII in comparison to the calculated NMR structure (blue helix-turn-helix motif). Based on the available information from NOESY spectra showing two sets of partially different NOEs and the specific selection of one state during the performed NMR titration experiments, we built approximate models of the assumed two individual states of the SrfTEII enzyme most likely representing an open (green) and a closed state (red). The two models differ in the helix-turn-helix motif. In the closed state, the helix-turn-helix motif (red) is showing an inward movement leading to a more compact structure in which the active site is less accessible. In the open state (green helix-turn-helix motif) the residues of the active site are less buried and become more accessible to the solvent, compared to the closed state and the calculated averaged structure represented as the structural bundle with the blue helixturn-helix motif. This opening also uncovers an interaction surface, which allows a tight complex formation with the misprimed T-domain. As discussed in the main text, these models are based on the assumption of only two different conformations and on titration-supported guesses which stretches of double resonances belong to which conformation. 9

10 Supplementary Figure 8 The interface between the T-domain and the SrfTEII shows a tight channel for accommodating the co-factor. A cut through the interface of the complex between the T-domain and the SrfTEII enzyme at the indicated position reveals a tight channel that is lined with the active site residues of the thioesterase. This channel is optimally suited for accommodating the modified co-factor and further emphasizes that large modifications of the co-factor (such as entire peptides) would lead to severe distortions of the tight interface between both domains. All structure presentations are generated using UCSF Chimera

11 Table 1 NMR and refinement statistics for the isolated SrfTEII NMR distance and dihedral constraints Distance constraints 2442 Total NOE 7802 Intra-residue 497 Inter-residue 1945 Sequential ( i-j = 1) 461 Medium-range (1< i-j < 5) 686 Long-range ( i-j 5) 798 Hydrogen bonds 92 dihedral angle constraints phi 139 psi 139 SrfTEII Structure statistics Violations (mean and s.d.) Distance constraints (Å) / Dihedral angle constraints ( ) / Max. dihedral angle violation ( ) 2.31 Max. distance constraint violation (Å) 0.21 CYANA target function value 4.9 Ų Average pairwise r.m.s.d (Å) 20 among 150 refined structures (residues 3-238) Heavy / Backbone /

12 Table 2 NMR and refinement statistics for the complex SrfTEII : TycC3-PCP SrfTEII TycC3-PCP NMR distance and dihedral constraints Distance restraints Total NOE Intra-residue Inter-residue Sequential ( i-j = 1) Non-sequential ( i-j > 1 ) Hydrogen bonds Protein protein intermolecular (H N (PCP):H FILV (TEII)) 23 Total dihedral angle constraints Protein phi psi Structure statistics Violations (mean and s.d.) Distance constraints (Å) / Dihedral angle constraints ( ) 3.4 +/- 1.2 Max. dihedral angle violation ( ) 4.8 Max. distance constraint violation (Å) 0.36 Average pairwise r.m.s.d.** (Å) 20 among 200 refined structures Protein complex Heavy / Backbone / Intermolecular interface / Complex Buried Surface Area (Ų) /- 280 intermolecular energies E tot (kcal/mol) /

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