Supporting Information

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1 Supporting Information Critical role of inter-domain interactions on the conformational change and catalytic mechanism of Endoplasmic Reticulum Aminopeptidase 1 Athanasios Stamogiannos 1, Zachary Maben 2, Athanasios Papakyriakou 1, Anastasia Mpakali 1, Paraskevi Kokkala 3, Dimitris Georgiadis 3, Lawrence J. Stern 2 and Efstratios Stratikos 1,* 1 National Centre for Scientific Research Demokritos, Agia Paraskevi, Athens, Greece 2 Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts, USA 3 Department of Chemistry, Laboratory of Organic Chemistry, University of Athens, Panepistimiopolis, Zografou, 15771, Athens, Greece Address correspondence to: Dr. Efstratios Stratikos, National Centre for Scientific Research Demokritos, Agia Paraskevi, Greece, stratikos@rrp.demokritos.gr or stratikos@gmail.com. Telephone: , Fax: S1

2 Supporting Table S1. Simulation systems of ligand-free ERAP1 employed for cmd and amd simulations and the corresponding range of interdomain angles (θ) sampled. Construct (sequence) wt-erap1 (33 941) 4mut-ERAP1 (33 941) Initial PDB ID_ Initial cmd State Chain θ (deg) (ns) Sampled amd Sampled θ (deg) (ns) θ (deg) Closed 2YD0_A MDJ_A Open 3MDJ_B MDJ_C Closed 2YD0_A MDJ_A Open 3MDJ_B MDJ_C S2

3 Supporting Table S2. MultiFoXS results for the two ERAP1 constructs. Multi-state models were fitted with q max = 0.26 Å -1 using all 3,222 ERAP1 conformations, which were extracted from the cmd trajectories after agglomerative clustering with a distance between clusters of 1.0 Å. The lowest χ and θ values for each ensemble size is given with the error indicating the range of χ values in the top 100 models, and the weighted average interdomain angle θ (± standard deviation) was calculated from the 100 top-scored (lowest-χ) ensembles. Ensemble size Lowest χ (error) KQ-ERAP1 θ (deg) Weighted average θ (deg) Lowest χ (error) 4mut-ERAP1 θ (deg) Weighted average θ (deg) (0.62) ± (0.46) ± (0.08) ± (0.06) ± (0.02) ± ((0.04) ± 0.7 S3

4 velocity (counts/(s*pmol)) WRVYEKC DNP ALK hydrolysis ERAP1 ERAP1 2mut ERAP1 4mut Supporting Figure S1: Rate of hydrolysis of the fluorigenic peptide WRVYEC(dnp)ALK by ERAP1 and ERAP1 variants 2mut and 4mut. S4

5 specific activity (pmol/(nmol*s)) ERAP1 ERAP1 2mut ERAP1 4mut w/o peptide QLESIINFEKL Supporting Figure S2: Hydrolysis of L-AMC by ERAP1 and variants was followed in the absence or presence of peptide QLESIINFEKL. The peptide inhibits L-AMC hydrolysis for wildtype ERAP1 but enhances hydrolysis rates for the two ERAP1 variants. S5

6 Specific activity (pmol/(h*ng)) Specific activity (pmol/(h*ng)) Specific activity (pmol/(h*ng)) Specific activity (pmol/(h*ng)) A B 60 LG10L hydrolysis 60 ERAP ERAP1 ERAP1 2mut ERAP1 4mut 0 LG3L LG4L LG5L LG6L LG7L LG8L LG9L LG10L C 8 ERAP1 2mut D 2.5 ERAP1 4mut LG3L LG4L LG5L LG6L LG7L LG8L LG9L LG10L 0.0 LG3L LG4L LG5L LG6L LG7L LG8L LG9L LG10L Supporting Figure S3: Panel A: Specific activity of ERAP1 and variants for the hydrolysis of the N-terminal Leucine residue of peptide LG10L (sequence LGGGGGGGGGGL). Panels B-D: Specific activity for the hydrolysis of the peptide LGnL peptide series by ERAP1 and variants. S6

7 S7

8 k obs (s -1 ) signal ERAP1-DG013A association nm nm nm 2000 nm time (s) B ERAP1-DG013A association 0.08 k on : 3.2*10-5 s -1 *nm k off : 11.0*10-4 s inhibitor concentration (nm) Supporting Figure S4: Panel A: Representative kinetic traces of L-AMC hydrolysis by ERAP1 after the rapid mixing of the indicated amounts of the DG013A inhibitor. Panel B: Linear relationship of the observed association rate of DG013A and ERAP1 and the concentration of the inhibitor. S8

9 K i app (nm) normalized activity normalized activity ERAP1 - DG013A (100 nm) association ERAP1 2mut - DG013A (700 nm) association 1.5 k obs : s k obs : s time (s) time (s) DG013A calculated dissociation constant ERAP1 ERAP1 2mut Supporting Figure S5: Top panels, 1 st order derivatives of kinetics of DG013A association with ERAP1 (left) and the ERAP1 variant 2mut (right). Red lines represent non-linear fits to a simple exponential decay function. Bottom, calculated dissociation constant of DG013A based on measured k on and k off values for DG013A and wild-type or 2mut ERAP1. S9

10 MVFLPLKWSL ATMSFLLSSL LALLTVSTPS WCQSTEASPK RSDGTPFPWN KIRLPEYVIP VHYDLLIHAN LTTLTFWGTT KVEITASQPT STIILHSHHL QISRATLRKG AGERLSEEPL QVLEHPRQEQ IALLAPEPLL VGLPYTVVIH YAGNLSETFH GFYKSTYRTK EGELRILAST QFEPTAARMA FPCFDEPAFK ASFSIKIRRE PRHLAISNMP LVKSVTVAEG LIEDHFDVTV KMSTYLVAFI ISDFESVSKI TKSGVKVSVY AVPDKINQAD YALDAAVTLL EFYEDYFSIP YPLPKQDLAA IPDFQSGAME NWGLTTYRES ALLFDAEKSS ASSKLGITMT VAHELAHQWF GNLVTMEWWN DLWLNEGFAK FMEFVSVSVT HPELKVGDYF FGKCFDAMEV DALNSSHPVS TPVENPAQIR EMFDDVSYDK GACILNMLRE YLSADAFKSG IVQYLQKHSY KNTKNEDLWD SMASICPTDG VKGMDGFCSR SQHSSSSSHW HQEGVDVKTM MNTWTLQKGF PLITITVRGR NVHMKQEHYM KGSDGAPDTG YLWHVPLTFI TSKSDMVHRF LLKTKTDVLI LPEEVEWIKF NVGMNGYYIV HYEDDGWDSL TGLLKGTHTA VSSNDRASLI NNAFQLVSIG KLSIEKALDL SLYLKHETEI MPVFQGLNEL IPMYKLMEKR DMNEVETQFK AFLIRLLRDL IDKQTWTDEG SVSERMLRSQ LLLLACVHNY QPCVQRAEGY FRKWKESNGN LSLPVDVTLA VFAVGAQSTE GWDFLYSKYQ FSLSSTEKSQ IEFALCRTQN KEKLQWLLDE SFKGDKIKTQ EFPQILTLIG RNPVGYPLAW QFLRKNWNKL VQKFELGSSS IAHMVMGTTN QFSTRTRLEE VKGFFSSLKE NGSQLRCVQQ TIETIEENIG WMDKNFDKIR VWLQSEKLER MHHHHHH Missing residues modeled/not modeled Not resolved in the open ERAP1 states Sequence conflicts/polymorphic sites His-tag added N-glycosylation Mutation sites Supporting Figure S6. Sequence of the ancestral human ERAP1 (isoform 1, UniProt entry Q9NZ08) employed for the wt-erap1 model with the R127P mutation. Polymorphic and N- glycosylation sites, missing residues in the X-ray crystal structures and the mutated residues in the 2mut/4mut-ERAP1 constructs are indicated and shown in Figure S7. S10

11 Supporting Figure S7. wt-erap1 model (3MDJ chain B) illustrating the domain organization, glycosylation sites, the 4 mutated residues, the modeled regions and the zinc-binding domain. S11

12 Supporting Figure S8. Side- and top-view animations of wt-erap1 (KQ-ERAP1 allele) illustrating the motions along the first two principal components PC-1 and PC-2, extracted from a total of 1.5 μs cmd simulations. Structures are color-coded from blue (positive) to red (negative) eigenvalues. The percent of the total variance of motions for each component is given in parenthesis, and the Latin numbers indicate the four domains. S12

13 Supporting Figure S9. Side- and top-view animations of 4mut-ERAP1 illustrating the motions along the first two principal components PC-1 and PC-2, extracted from a total of 1.5 μs cmd simulations. Structures are color-coded from blue (positive) to red (negative) eigenvalues. The percent of the total variance of motions for each component is given in parenthesis, and the Latin numbers indicate the four functional domains. S13

14 Supporting Figure S10. Plots of the interdomain angle θ (deg) as a function of simulation time (excluding the initial 10-ns equilibration period) from the cmd simulations of wt-erap1 (indicated as KQ-ERAP1, upper panel) and 4mut-ERAP1 (lower panel) in the open state. The panels on the right show the probability density distributions of θ obtained using the kernel density estimator (Gaussian window) of the R package. [1] The three initial models of open ERAP1 were based on the three monomers in the asymmetric unit of the PDB ID: 3MDJ and are color-coded per chain using black lines for A, red for B and blue for C. 1 R Development Core Team (2008). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN , S14

15 Supporting Figure S11. Distribution of the interdomain angle θ (upper plot) and dihedral angle φ (lower plot) obtained from the aggregate of 1.5 μs cmd simulations of wt-erap1 (KQ- ERAP1, black lines) and 4mut-ERAP1 (red lines) in the open states (results of the ns shown in Figure S10). S15

16 Supporting Figure S12. Interdomain angle θ (deg) of wt-erap1 as a function of amd simulation time (in thousands of 2-fs steps). The ten independent amd simulations that were seeded from the cmd simulations are color-coded by the initial θ value and split into two graphs for clarity. S16

17 Supporting Figure S13. Interdomain angle θ (deg) of 4mut-ERAP1 as a function of amd simulation time (in thousands of 2-fs steps). The ten independent amd simulations that were seeded from the cmd simulations are color-coded by the initial θ value and split into two graphs for clarity. S17

18 Supporting Figure S14. Density distributions of the interdomain angle θ of wt-erap1 (upper graph) and 4mut-ERAP1 (lower graph) as a function of the number of amd simulations used for processing. Ten independent amd simulations of 50 million 2-fs steps were seeded from the cmd simulations at variable initial angle θ between deg (Figures S12, S13). The 6 amd sets correspond to those initiated at θ = deg and the 8 amd sets are for initial θ = deg. S18

19 Supporting Figure S15. FoXS results displaying the fit of wt-erap1 and 4mut-ERAP1 experimental profiles using the X-ray structures (A, B) and the initial molecular models used for the MD calculations (C, D). The crystallographic structures of ERAP1 in the open state are chains A, B and C of PDB ID: 3MDJ and ERAP1 in the closed state PDB ID: 2YD0. S19