Tu 1,*, , Sweden

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1 Supplementary Material Computational studiess of the binding profile of phosphoinositide PtdIns(,4,5)P with the pleckstrin homology domain d of an oomycetee cellulose synthase Guanglin Kuang 1, Vincent Bulone 2, and Yaoquan T Tu 1, 1 Division of Theoretical Chemistry and Biology, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University y Center, Stockholm, 10691, Sweden 2 Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNovaa University Center, Stockholm,, , Sweden Supplementary Figure S1. Ramachandrann plot of the homologyy model of SmCesA2-PH.

2 Supplementary Figure S2. Dynamic behaviors of SmCesA2-PH in two independent MD simulations with the Amber99SB force field. (a, c) RMSD plots of the backbone atoms of the -sandwichh and -helix core structure (black), VL1 (red) and VL2 (green) off SmCesA2-PH. (b, d) The RMSF plot of SmCesA2-PH. Supplementary Figure S. Binding mode of Ins(,4,5)P with the template structure TAPP1-PH.

3 Supplementary Figure S4. Some MD plots of the head groupss used to monitor their binding processes with SmCesA2-PH in i the MD simulations with different initial velocities (second run). COM distance is the distance betweenn the centers of mass (COMs) of the head group and the protein. RMSD is the root mean square deviation of the head group conformation in the MD simulations after the alignment of the protein. Minimumm distance is the minimum distance between the head group and protein heavy atoms. Supplementary Table S1. Schrödinger XP docking scores off Ins(,4,5)PP with the mutated forms of the hot-spot residues identified by molecular docking and molecular dynamics simulations. wild type K85A K88A K100A R102A Glide XP Docking Score (kcal/mol)

4 Supplementary Figure S5. (a) Binding mode of Ins(,4,5)P in i a meta-stable state.(b) Some parameters to monitor the binding profilee of the meta-stable state. COM distance is the t distance between the centers of mass (COMs) of thee inositol head group and the protein. RMSD is the root mean square deviation of the head group conformationn in the MD simulation n after the alignment of the protein. Minimumm distance iss the minimum distance between the t head group and protein heavy atoms. Dihedral is the same as that t definedd in the Method section.

5 Supplementary Figure S6. (a) Bindingg mode of SmCesA2-PH with h the PtdIns(,4,5)P molecule in solution. (b) Some parameters used to monitor the binding profile in i the MD simulations. COM distance is the distance between the centers of mass m (COMs) of the inositol head group and the protein. Head RMSD is the RMSD of the inositol head group. Tail RMSD is the RMSD of the two hydrophobic tails. Minimum distance is the minimum distance between the lipid molecule and protein heavy atoms.

6 Supplementary Figure S7. Binding profile of SmCesA2-PHH on the PtdIns(,4,5)P /POPC membranee in two independent MD simulations. The figures show the plotss of the RMSDs of the backbone atoms of the -sandwich and -helix core structure (RMSD1, black), VL1 (RMSD2, blue) and VL2 (RMSD, cyan) as well as thee minimumm distance between SmCesA2-PH and the PtdIns(,4,,5)P /POPC membranee (green). a b

7 c d Supplementary Figure S8. Binding profilee of SmCesA2-PH with PtdIns(,4)P 2. (a) The binding mode of SmCesA2-PH with the head groupp Ins(,4)P 2. (b) Somee parameters used to monitor the binding profile of the head group in MD simulations. COM distance is thee distance between the centers of mass (COMs) of the inositol headd group and the protein. RMSD iss the root mean square deviation of the head group conformation inn the MD simulation after a the alignment of the t protein. Minimum distance is the minimum distancee between the head group and protein heavy atoms. (c) The binding profile of SmCesA2-PH with the PtdIns( (,4)P 2 /POPC membrane which includes the contact (within Å) probability plots of the amino acids of SmCesA2-PH with the PtdIns(,4) )P 2 /POPC membrane, and the binding mode of SmCesA2-PH with the membrane. (d)

8 The figures showing the plots of the RMSDs of the backbone atoms of the -sandwich and -helixx core structure (RMSD1, black),, VL1 (RMSD2, blue) and VL2 (RMSD, cyan) as well as the minimum distance between SmCesA2-PH and the PtdIns(,4)P 2 /POPC membrane (green). Supplementary Figure S9. Binding profile of SmCesA2-PH on the POPC membrane in two independent MD simulations. The figures show the plots of the RMSDs R of the backbone atoms of the -sandwich and -helix core structure (RMSD1, black), VL1 (RMSD2, blue) and VL2 (RMSD, cyan) as well as the minimum distance between SmCesA2-PH andd the POPC membrane (green). Supplementary Figure S10. Binding profile of SmCesA2-PH on o the PtdIns/POPC membranee in two independent MD simulations. The figures show the plots of the RMSDs of the backbone atoms of the -sandwich and -helix core structure (RMSD1, black), VL1 (RMSD2, blue) and VL2 (RMSD, cyan) as well as the minimum distance between SmCesA2-PH and the PtdIns/POPC membrane.

9 Supplementary Figure S11. metadynamics simulations of Illustration of the collective variables selected for the the SmCesA2-Ins complex. CV1 is the distance d between the center of mass (COM) of the ligand and thatt of SmCesA2-PH. CV2 is the dihedral angle (torsion ) ) defined by the two end points of the -helix,, the COM of the protein, and the COM of the ligand. Supplementary Figure S12. Evolutions off COM distance (CV1) and Gaussian HILLS height in the metadynamics simulations. Several recrossing events of the distance CV and smalll Gaussian HILLS height (close to zero) are two common indicators of the convergence of the

10 metadynamics simulations. Thesee plots indicate that the three systems have converged. Supplementary Figure S1. Evolutions of the ALPHARMSD in the three metadynamics simulations, which can be used to monitorr the conten of -helix. Supplementary Figure S14. Initial setup of the SmCesA2-PH are shown in the thick stick SmCesA2-PH and PtdIns(,4,5)P /POPC membranee system. The PtdIns(,4,5)P molecule and three critical residuess in the binding site of mode and the POPC molecules m in the thin stick s mode. The protein is shown in the cartoon mode. Supplementary Videos Supplementary Video S1. Trajectory of SmCesA2-PH in a 500 ns MD simulation. Supplementary Video S2. Binding profilee of SmCesA2-PH with the PtdIns(,4,5)P moleculee in solution in a 500 ns MD simulation.

11 Supplementary Video S. Binding profile of SmCesA2-PH on the PtdIns(,4,5)P /POPC membrane in a 500 ns MD simulation. Supplementary Video S4. Binding profile of SmCesA2-PH on the PtdIns(,4)P 2 /POPC membrane in a 500 ns MD simulation. Supplementary Video S5. Binding profile of SmCesA2-PH on the POPC membrane in a 500 ns MD simulation. Supplementary Video S6. Binding profile of SmCesA2-PH on the PtdIns/POPC membrane in a 500 ns MD simulation. Appendix S1 Force field parameters of the head groups of phosphoinositides, obtained from CHARMM ParamChem web server Ins Toppar stream file generated by CHARMM General Force Field (CGenFF) program version beta For use with CGenFF version 2b8 read rtf card append Topologies generated by CHARMM General Force Field (CGenFF) program version beta 6 1! "penalty" is the highest penalty score of the associated parameters.! Penalties lower than 10 indicate the analogy is fair; penalties between 10! and 50 mean some basic validation is recommended; penalties higher than! 50 indicate poor analogy and mandate extensive validation/optimization. RESI INS GROUP! param penalty= ; charge penalty=! CHARGE CH_PENALTY ATOM O2 ATOM O ATOM O4 ATOM O5 ATOM O6 ATOM C ! ATOM C ! ATOM O12 ATOM C ! ATOM C ! ATOM C15 ATOM C ! 0.146!

12 ATOM H1 ATOM H2 ATOM H ATOM H4 ATOM H5 ATOM H6 ATOM H7 ATOM H8 ATOM H9 ATOM H10 ATOM H11 ATOM H12 BOND O2 BOND O BOND O4 BOND O5 BOND O6 BOND C1 BOND O2 BOND O BOND O4 BOND O5 BOND O6 BOND O12 BOND C1 BOND C16 C12 C1 C14 C15 C16 C16 C12 O12 C1 C14 C15 C16 H1 H2 H H4 H5 H6 H7 H8 H9 H10 H11 H12 END read param card flex append Parameters generated by analogy by CHARMM General Force Field (CGenFF) program version beta! Penalties lower than 10 indicate the analogy is fair; penalties between 10! and 50 mean some basic validation is recommended; penalties higher than! 50 indicate poor analogy and mandate extensive validation/optimization. BONDS ANGLES DIHEDRALS ! INS, from CG21, penalty= ! INS, from CG21, penalty= 4 IMPROPERS END RETURN Ins(1)P (PIP) Toppar stream file generated by CHARMM General Force Field (CGenFF) program version beta For use with CGenFF version 2b8

13 read rtf card append Topologies generated by CHARMM General Force Field (CGenFF) program version beta 6 1! "penalty" is the highest penalty score of the associated parameters.! Penalties lower than 10 indicate the analogy is fair; penalties between 10! and 50 mean some basic validation is recommended; penalties higher than! 50 indicate poor analogy and mandate extensive validation/optimization. RESI PIP 2.000! param penalty= ; charge penalty= 2.12 GROUP! CHARGE CH_PENALTY ATOM C11 ATOM C ! 0.146! ATOM C ! ATOM C14 ATOM C ! 0.098! ATOM C ! 2.12 ATOM O5 ATOM P5 0.99! 1.099! ATOM OP ! ATOM OP5 ATOM OP ! 0.900! ATOM O ATOM O6 ATOM O 0.00 ATOM O12 ATOM O2 ATOM H1 ATOM H2 ATOM H ATOM H ATOM H5 ATOM H6 ATOM H ATOM H8 ATOM H9 ATOM H10 ATOM H11 BOND C1 BOND C1 BOND C16 BOND O5 BOND P5 BOND P5 BOND P5 BOND C1 BOND C16 BOND O4 BOND O6 BOND O BOND O12 BOND O2 C16 O12 C12 O2 C1 C14 O O4 C15 O5 C16 O6 P5 OP4 OP6 OP5 H1 H2 H H4 H5 H6 H7 H8 H9 H10 H11 END read param card flex append

14 Parameters generated by analogy by CHARMM General Force Field (CGenFF) program version beta! Penalties lower than 10 indicate the analogy is fair; penalties between 10! and 50 mean some basic validation is recommended; penalties higher than! 50 indicate poor analogy and mandate extensive validation/optimization. BONDS ANGLES ! PIP, from CG1, penalty= 1.5 DIHEDRALS ! PIP, from CG21, penalty= ! PIP, from CG21 CG21, penalty= ! PIP, from CG21, penalty= ! PIP, from CG21, penalty= ! PIP, from CG21, penalty= ! PIP, from CG1, penalty= ! PIP, from CG1, penalty= ! PIP, from CG1, penalty= 1.5 IMPROPERS END RETURN Ins(,4)P 2 (PIP2) Toppar stream file generated by CHARMM General Force Field (CGenFF) program version beta For use with CGenFF version 2b8 read rtf card append Topologies generated by CHARMM General Force Field (CGenFF) program version beta 6 1! "penalty" is the highest penalty score of the associated parameters.! Penalties lower than 10 indicate the analogy is fair; penalties between 10! and 50 mean some basic validation is recommended; penalties higher than! 50 indicate poor analogy and mandate extensive validation/optimization. RESI PIP2 GROUP.000! param penalty= ; charge penalty=! CHARGE CH_PENALTY ATOM C ! ATOM C12 ATOM C ! 0.094! ATOM C ! ATOM C15 ATOM C ! 0.146! ATOM O 0.99! ATOM P ATOM O 1.099! 0.900! ATOM O ! ATOM O1 ATOM O ! 0.00 ATOM O ! ATOM P4 ATOM O ! 0.670! ATOM O ! 0.00 ATOM O4 ATOM O ! 0.00 ATOM O ATOM O6 ATOM H11

15 ATOM H ATOM H1 ATOM H ATOM H ATOM H16 ATOM HO2 ATOM HO ! ATOM HO12 ATOM HO5 ATOM HO6 BOND C1 BOND C1 BOND C1 BOND C16 BOND C16 BOND O BOND P BOND P BOND P BOND O2 BOND O4 BOND P4 BOND P4 BOND P4 BOND O42 BOND O12 BOND O5 BOND O6 C12 C16 O12 H11 C1 O2 H12 C14 O H1 C15 O4 H14 C16 O5 H15 O6 H16 P O O2 O1 HO2 P4 O42 O41 O4 HO42 HO12 HO5 HO6 END read param card flex append Parameters generated by analogy by CHARMM General Force Field (CGenFF) program version beta! Penalties lower than 10 indicate the analogy is fair; penalties between 10! and 50 mean some basic validation is recommended; penalties higher than! 50 indicate poor analogy and mandate extensive validation/optimization. BONDS ANGLES ! PIP2, from CG1, penalty= ! PIP2, from CG21, penalty= 0.6 DIHEDRALS ! PIP2, from CG21, penalty= ! PIP2, from CG21 CG21, penalty= ! PIP2, from OG02 CG21, penalty= ! PIP2, from CG21, penalty= ! PIP2, from CG21, penalty= ! PIP2, from CG21, penalty= ! PIP2, from CG1, penalty= ! PIP2, from CG1, penalty= ! PIP2, from CG1, penalty= ! PIP2, from CG1, penalty= ! PIP2, from CG1, penalty= 1.5

16 ! PIP2, from CG1, penalty= ! PIP2, from, penalty= ! PIP2, from CG21, penalty= ! PIP2, from CG21, penalty= ! PIP2, from CG21, penalty= 0.6 IMPROPERS END RETURN Ins(,4,5)P (PIP) Toppar stream file generated by CHARMM General Force Field (CGenFF) program version beta For use with CGenFF version 2b8 read rtf card append Topologies generated by CHARMM General Force Field (CGenFF) program version beta 6 1! "penalty" is the highest penalty score of the associated parameters.! Penalties lower than 10 indicate the analogy is fair; penalties between 10! and 50 mean some basic validation is recommended; penalties higher than! 50 indicate poor analogy and mandate extensive validation/optimization. RESI PIP 5.000! param penalty= ; charge penalty=.268 GROUP! CHARGE CH_PENALTY ATOM P ATOM O ! 0.82! ATOM O2 0.82! 0.00 ATOM O ATOM C ! 0.00! ATOM C ! ATOM C11 ATOM C ! 0.150! ATOM C !.22 ATOM C14 ATOM O ATOM O12 ATOM O6 ATOM O5 0.99! ATOM P ! ATOM O51 ATOM O ! 0.900! ATOM O ! ATOM O4 ATOM P4 0.99! 1.099! ATOM O ! ATOM O4 ATOM O ! 0.900! ATOM O 0.670! ATOM H1 ATOM H ATOM H11 ATOM H16 ATOM H ATOM H ATOM HO2 ATOM HO12 ATOM HO6 ATOM HO 0.41! BOND P BOND P BOND P O1 O2 O

17 BOND P BOND O BOND C1 BOND C1 BOND C1 BOND C16 BOND C16 BOND C16 BOND O2 BOND O12 BOND O6 BOND O5 BOND P5 BOND P5 BOND P5 BOND O4 BOND P4 BOND P4 BOND P4 BOND O O C1 C12 C14 H1 C11 O2 H12 C16 O12 H11 C15 O6 H16 C14 O5 H15 O4 H14 HO2 HO12 HO6 P5 O51 O5 O52 P4 O41 O4 O42 HO END read param card flex append Parameters generated by analogy by CHARMM General Force Field (CGenFF) program version beta! Penalties lower than 10 indicate the analogy is fair; penalties between 10! and 50 mean some basic validation is recommended; penalties higher than! 50 indicate poor analogy and mandate extensive validation/optimization. BONDS ANGLES ! PIP, from CG1, penalty= ! PIP, from CG21, penalty= 0.6 DIHEDRALS ! PIP, from CG21, penalty= ! PIP, from CG21 CG21, penalty= ! PIP, from OG02 CG21, penalty= ! PIP, from CG21, penalty= ! PIP, from CG21, penalty= ! PIP, from CG21, penalty= ! PIP, from CG1, penalty= ! PIP, from CG1, penalty= ! PIP, from CG1, penalty= ! PIP, from CG1, penalty= ! PIP, from CG1, penalty= ! PIP, from CG1, penalty= ! PIP, from, penalty= ! PIP, from CG21, penalty= ! PIP, from CG21, penalty= ! PIP, from CG21, penalty= 0.6 IMPROPERS END RETURN