Supplementry Informtion Coopertion of locl motions in the Hsp90 moleculr chperone ATPse mechnism Andre Schulze 1, Gerti Beliu 1, Dominic A. Helmerich 1, Jonthn Schubert 1, Lurence H. Perl 2, Chrisostomos Prodromou 2 & Hnnes Neuweiler 1, * 1 Deprtment of Biotechnology nd Biophysics, Julius-Mximilins-University Würzburg, Am Hublnd, 97074 Würzburg, Germny 2 Genome Dmge nd Stbility Centre, School of Life Sciences, University of Sussex, Flmer, Brighton BN1 9RQ, United Kingdom *Corresponding uthor: hnnes.neuweiler@uni-wuerzburg.de 1
Supplementry Tbles Supplementry Results Tble 1: Kinetic prmeters of locl motions of Hsp90 mesured t 25 C. Mutnt Reporter 1 k1 2 k 2 3 k 3 men k b Wild type β-strnd swp 0.59 ±0.07 0.31 0.19 0.05 ±0.005 0.22 1.47 ±0.24 0.18 Wild type ssocition 0.68 ±0.05 0.41 0.32 ±0.008 0.08 ±0.007 0.18 Wild type Lid closure 0.55 0.33 0.45 ±0.007 0.10 ±0.002 0.16 ±0.003 A2W-L160W β-strnd swp c 0.35 ±0.07 0.10 0.65 ±0.04 0.02 ±0.001 0.03 ±0,0005 A2W-L160W ssocition 0.72 ±0.04 0.20 0.28 ±0.04 0.04 ±0.004 0.10 ± 0.01 A2W-L160W Lid closure 0.5 0.14 0.5 ±0.07 0.05 ±0.002 0.07 ±0.0005 A107N β-strnd swp 0.61 ±0.05 2.47 ±0.37 0.27 ±0.05 0.62 ±0.10 0.12 0.05 0.34 A107N ssocition 0.84 A107N Lid closure 0.32 ±0.09 1.63 ±0.05 0.40 ±0.08 0.16 0.68 ±0.14 0.20 ±0.04 1.37 ±0.21 0.78 ±0.06 0.79 ±0.08 n nd kn re the reltive fluorescence mplitudes nd observed rte constnts (1/ n in 1/min). Dt represent men vlues ± s.d. of three mesurements. b The men rte constnt ws clculted from the sum of individul time constnts weighted by the respective mplitudes (1/(Σn n). c To monitor domin swp of the TrpZip mutnt A2W-L160W, the fluorophore ws plced in witing position on the second subunit of heterodimeric Hsp90 (E162C). Tble 2: ATPse ctivity of wild-type Hsp90 nd mutnts thereof mesured t 37 C. Mutnt ATPse (min -1 ) Wild type 1.6 ± 0.04 A2W-L160W 0.61 ± 0.09 T101I 0.35 ± 0.06 F349A 0.12 ± 0.02 R380A ND ND; not detectble; dt represent men vlues ± s.d. of three mesurements. 2
Tble 3: Kinetics of locl motions of wild-type Hsp90 in the presence of Ah1 mesured t 25 C. Mutnt Reporter + Ah1 1 k1 2 k 2 men k b Wild type β-strnd swp 0.59±0.06 35.3±3.0 0.41±0.06 1.8±0.27 4.2±0.25 Wild type ssocition 0.80 18.4±0.97 0.20 2.1 7.2±0.5 Wild type Lid closure 0.52±0.04 10.5±2.2 0.48±0.04 2.1±0.4 3.6±0.55 F349A β-strnd swp 0.62±0.08 2.84±0.22 0.28±0.1 0.49±0.002 0.1 0.05 0.31±0.06 F349A ssocition 0.70±0.15 0.94±0.41 0.3±0.07 0.15 0.35±0.08 F349A Lid closure ND ND ND ND ND n nd kn re the reltive fluorescence mplitudes nd observed rte constnts (1/ n in 1/min). Dt represent men vlues ± s.d. of three mesurements. b The men rte constnt ws clculted from the sum of individul time constnts weighted by the respective mplitudes (1/(Σn n). ND; not detectble. 3
Tble 4: Dynmics of NTD structurl elements mesured by PET-FCS t 25 C N-terminl domin 1 1 (µs) 2 2 (µs) 3 3 (µs) Q14C-A2W 0.67±0.06 392±23 0.10 6±3 -- -- Q14C-A2W +ATP 0.53 429±36 0.08 11±10 Q14C-A2W-L160W -- -- -- -- -- -- A112C-S25W 0.78 375±18 0.14±01 7±2 -- -- A112C-S25W + ATP 0.29 300±4 0.15 7±1 NM-domin Q14C-A2W 1.03±0.09 940±140 0.59±0.12 145±38 0.15 0.9±0.8 Q14C-A2W +ATP 0.89 882±79 0.42±0.06 130±24 0.18±0.05 0.3±0.1 Q14C-A2W +Ah1 0.92 1000±100 0.61±0.07 170±20 0.15±0.08 1.9±0.8 A112C-S25W 0.56 350±40 0.26 15±7 0.27±0.05 0.5±0.4 A112C-S25W + ATP 0.20 233±13 0.19 11±1 0.21±0.05 0.2±0.1 A112C-S25W + Ah1 0.17 157±20 0.20 7±1 0.45±0.14 0.2±0.1 n nd n re the observed mplitudes nd corresponding time constnts. Dt represent men vlues ± s.d. of three mesurements. 4
Supplementry Figures Figure 1 Figure 1: Influence of ADP on conformtionl switches of Hsp90 Fluorescence intensity time trces mesured for () -strnd swp (A2C+E162W), (b) -domin ssocition (E192C-N298W), nd (c)/(d) lid closure (reporters S51C-A110W nd S51W-A110C, respectively). 4 mm ADP ws dded t time t = 0 min. Mesurements were done t 25 C. 5
Figure 2 Figure 2: Kinetics of erly remodeling of the lid induced by binding of nucleotide () Representtive fluorescence intensity time trces of reporter S51W-A110C on Hsp90 mesured using stopped-flow fluorescence spectroscopy. Kinetic trnsients were triggered by binding of 0.5 mm ADP (green) nd 0.75 mm ADP (blue). Ech time trce is n verge over three individul shots. Blck lines re single exponentil fits to the dt. We used ADP insted of ATP to solely trigger remodeling of the lid nd to void possible interference from other conformtionl chnges tht could complicte dt nlysis. (b) Observed rte constnts plotted versus concentrtion of ADP. Dt represent men vlues ± s.d. of three mesurements (performed t 25 C). The blck line is liner fit to the dt tht yielded bi-moleculr rte constnt of 1.3±0.2 x 10 5 M -1 s -1. Figure 3 Figure 3: Heterogeneity of kinetics of conformtionl switching Plot of the observed mplitudes nd rte constnts of the individul exponentil phses in multi-exponentil decys of conformtionl switching (see Supplementry Tble 1). Amplitudes (filled brs) nd corresponding rte constnts (ptterned brs) re shown in sme color. Pnels (), (b), nd (c), show kinetics mesured for wild-type Hsp90, the TrpZip construct, nd mutnt A107N, respectively. Dt represent men vlues ± s.d. of three mesurements. 6
Figure 4 Figure 4: Influence of binding of AMP-PNP on motion of the lid in isolted NTD nd NMdomin. () Fluorescence intensity time trces mesured for reporter A110C-S51W on the NTD in isoltion, which probes closure of the lid. 2 mm AMP-PNP ws dded t time t = 0 min. (b) The sme mesurement for reporter S51C-A110W on the NM-domin in bsence (blck) nd presence (blue) of 20 µm Ah1. Ah1 ws dded t t = -10 min nd AMP-PNP t t = 0 min. Mesurements were done t 25 C. 7
Figure 5 Figure 5: Anlysis of PET-FCS dt using model tht lcks 300-900 µs exponentil relxtion. (), (b) ACFs recorded from -strnd reporter Q14C-A2W nd from lid reporter A112C-S25W, respectively, on the isolted NTD. (c), (d) ACFs recorded from -strnd reporter Q14C-A2W nd from lid reporter A112C- S25W, respectively, on the NM-domin. In cse of the NTD (() nd (b)), cyn lines re fits to the dt using model for moleculr diffusion contining two single-exponentil relxtions. In cse of the NM-domin ((c) nd (d)), cyn lines re fits to the dt using model for moleculr diffusion contining three singleexponentil relxtions. Red lines re fits to the dt using the sme models but lcking single-exponentil relxtion t 300-900 µs. These fits yielded rtificilly smll diffusion time constnts due to overlp of time scles of diffusion nd conformtionl fluctutions. Specificlly, the NTD hd diffusion time constnt of 1.7 ms tht ws rtificilly reduced to 0.6-0.7 ms. The NM-domin hd diffusion time constnt of 2.2 ms tht ws rtificilly reduced to 1.9 nd 0.9 ms. 8