Native Hydrophobic Binding Interactions at the Transition State for

Subscriber access provided by FLORIDA ATLANTIC UNIV

Article

Native Hydrophobic Binding Interactions at the Transition State for Association between the TAZ1 Domain of CBP and the Disordered TAD-STAT2 are not a Requirement Ida Lindström, and Jakob Dogan Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00428 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 18, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Native Hydrophobic Binding Interactions at the Transition State for Association between the TAZ1 Domain of CBP and the Disordered TAD-STAT2 are not a Requirement

Ida Lindström1 and Jakob Dogan1* 1

Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden * Corresponding author: e-mail: [email protected] Telephone: 46-8-162470

1

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABBREVIATIONS TAZ1, transcriptional adaptor zinc-binding 1; CBP, CREB binding protein; TAD-STAT2, transactivation domain of the signal transducer and activator of transcription 2; TCEP, Tris(2carboxyethyl)phosphine; CD, circular dichroism; ITC, isothermal titration calorimetry

2


Page 2 of 28

Page 3 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

ABSTRACT A significant fraction of the eukaryotic proteome consists of proteins that are either partially or completely disordered in native-like conditions. Intrinsically disordered proteins (IDPs) are common in protein-protein interactions and are involved in numerous cellular processes. Although many proteins have been identified as disordered, much less is known about the binding mechanisms of the coupled binding and folding reactions involving IDPs. Here we have analyzed the rate-limiting transition state for the binding between the TAZ1 domain of CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2) by site-directed mutagenesis and kinetic experiments (Φ-value analysis), and found that the native protein-protein binding interface is not formed at the transition state for binding. Instead, native hydrophobic binding interactions form late, after crossing the rate-limiting barrier. The association rate constant in the absence of electrostatic enhancement was determined to be rather high which is consistent with the Φ-value analysis that there are few or no obligatory native contacts. Also, linear free energy relationships clearly demonstrate that native interactions are cooperatively formed, a scenario that has been usually observed for proteins that fold according to the so-called nucleation–condensation mechanism. Thus, native hydrophobic binding interactions at the rate-limiting transition state for association between TAD-STAT2 and TAZ1 are not a requirement, which is generally in agreement with previous findings on other IDP systems and might be a common mechanism for IDPs.

3


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 28

INTRODUCTION Many proteins need to be folded into a specific three-dimensional structure in order to carry out their function, for instance, by binding to a certain target. However, it has been recognized for some time that a significant fraction of the eukaryotic proteome consists of so-called intrinsically disordered proteins (IDPs), many of which undergo a disorder-to-order transition upon binding their partner1-5. IDPs have crucial roles in various cellular processes, for example, in signaling and regulation6, but many are also associated with diseases such as cancer and neurodegenerative disorders7. Due to the abundance and functional importance of IDPs, there have been a great interest to better understand how IDPs bind to their targets, what the binding mechanisms are, and the general advantages that disorder may provide in protein-protein interactions6, 8-10. IDPs have therefore been the subject of intense research the last couple of years, and the majority of these studies have been computational or been carried out at equilibrium, which have provided important insights on the relationships between structure, dynamics and function6. However, very few experimental investigations on the kinetics have been reported11-15, and such studies are essential in order to determine the binding mechanisms of the coupled binding and folding of IDPs16, 17.

CREB binding protein (CBP) is a multidomain transcriptional co-activator that is involved in the regulation and activation of gene expression2, 18, 19. The transcriptional adaptor zinc-binding 1 (TAZ1) is a globular helical domain of CBP that mediates interactions with a number of different proteins, many of which are disordered, including the transactivation domain of the signal transducer and activator of transcription 2 (TAD-STAT2)6,

20, 21

. STAT2 in the JAK-STAT

pathway play an important role in cytokine-induced signal transduction from the cell membrane

4


Page 5 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

to the nucleus, resulting in activation of genes22. TAD-STAT2 undergoes a disorder-to-order transition upon binding TAZ123. The solution three-dimensional NMR structure of the TAZ1/TAD-STAT2 complex has been previously determined, which shows that TAD-STAT2 has a rather extended conformation, forming helical segments and wraps around TAZ1 (Figure 1), resulting in a large interaction surface which consists of a hydrophobic core and electrostatic interactions23.

Figure 1. Structure of the TAZ1/TAD-STAT2 complex (PDB code 2KA4)23. TAZ1 is shown in gray, with the side chain of Trp-418 in TAZ1 shown in green, and TAD-STAT2 is shown in pink. Trp-418 was used as a fluorescence probe in this study. Image was generated using PyMol.

5


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

To elucidate the molecular details of the coupled binding and folding reaction of TAZ1 and TAD-STAT2 we have in this work used protein engineering together with kinetic experiments to determine the transition state structure in terms of formation of native contacts in the binding interface. Our study demonstrates that very little native hydrophobic interactions are formed in the rate-limiting transition state for binding, and are instead formed in a cooperative manner after the rate-limiting barrier for association. The implications of our results in the light of previous studies on IDPs are discussed.

MATERIAL AND METHODS Protein expression and purification: Human TAZ1 (residues 340-439), and TAD-STAT2 (residues 786-838, C793H/C809S) DNA sequences were purchased from Genscript and inserted into a modified pRSET vector (Thermo Fischer) resulting in a final construct of His6-Lipoyl domain-thrombin site-[TAZ1 or TAD-STAT2]. The protein sequences used in this study are the same as those used in the 3D structure determination of the TAZ1/TAD-STAT2 complex by Wright and colleagues23. The plasmid was transformed into Escherichia coli BL21 pLysS cells (Thermo Fischer) and grown in 2xTY-media at 37 ˚C until an OD600 of 0.6-0.7 was reached, and then induced with 0.5 mM and 0.8 mM isopropyl β-D-thiogalactopyranoside to overexpress TAD-STAT2 and TAZ1, respectively. Also, 150 µM ZnSO4 was added to the TAZ1 culture at the time of induction as previously described24. The cultures were then cultivated for an additional 12-16 hours at 15 °C for TAZ1 or at 18 °C for TAD-STAT2. Cells were lysed by sonication and the cell debris was removed by centrifugation at 4 °C. The lysate was sterile filtered and loaded onto a nickel-sepharose 6 fast flow (GE Healthcare) column. After washing the column with 20 mM Tris (pH=8.0 for TAD-STAT2 and pH=7.7 for TAZ1), 500 mM NaCl,

6


Page 6 of 28

Page 7 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

5-10 mM imidazole, the fusion protein was eluted using 250 mM imidazole, 500 mM NaCl (1 mM TCEP was added to the buffers for TAZ1). The protein solution was dialyzed against 20 mM Tris (pH=8.0 for TAD-STAT2, and pH=7.5 for TAZ1), 100 mM NaCl (TAD-STAT2) or 150 mM NaCl (TAZ1), and 1 mM TCEP (only for TAZ1), at 4 °C, after which thrombin (GE Healthcare) was added to cleave the tag off. The NaCl concentration for the TAZ1 solution was adjusted to 600 mM and then loaded onto a benzamidine column to remove the thrombin, after which 6-6.5 M urea was added to the TAZ1 solution. The solution was once again loaded onto the Ni-column to separate the His6-Lipoyl domain from TAZ1 and to remove other impurities. The TAD-STAT2 solution was loaded onto a Ni-column with a serially attached benzamidine column to remove the tag and other impurities, as well as thrombin. As a final purification step all proteins were subjected to a reversed phase (RP) chromatography purification step using a resource (GE Healthcare) RP column (standard H2O/acetonitrile solvents, 0.1% (v/v) TFA). Mutant variants were generated by the quick-change approach using pfu Ultra DNA polymerase, and DpnI, and expressed and purified as described above. TAZ1 was refolded by the addition of 3:1 (Zn:TAZ1) molar ratio of ZnSO4 as previously described24 and monitored by circular dichroism spectroscopy.

Stopped-flow fluorimetry: The binding kinetics was investigated using a SX-18MV stopped-flow spectrometer (Applied photophysics, Leatherhead, U.K.). Measurements were carried out in 20 mM HEPES, 190 mM NaCl, 1 mM TCEP, pH 6.9, and at 293 K. For the ionic strength dependence study, the NaCl concentration was adjusted in to order to obtain the desired ionic strength (I), from I=0.04 M to 1 M. There is a single tryptophan in TAZ1 (Figure 1) that was used as a fluorescence probe. Excitation was at 295 nm, and the fluorescence change was followed using a 320 nm long pass filter. The concentration of TAZ1 was held constant at 0.6-0.9 µM 7


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

while the concentrations of the TAD-STAT2 variants were varied. The observed rate constants, kobs,, were extracted from the binding traces, which were observed to be biphasic for TADSTAT2 and its mutant variants, with a fast and a slow phase. The kobs for the fast phase is linearly dependent on TAD-STAT2 concentration, and the kobs for the slow phase is concentration independent in the concentration range where we have collected the data. The possibility that the slow phase could be due to an artifact was discarded since the slow phase was absent when mixing the molecule N-acetyl-L-tryptophanamide (NATA) with buffer, and it was also not present when mixing TAZ1 with buffer. The apparent association rate constant (konapp) was

determined by fitting the concentration dependence of kobs for the fast phase to the general equation for the reversible association of two molecules25. Datafitting was performed using KaleidaGraph (Synergy Software). The kobs for the slow phase for TAD-STAT2 and all its mutant variants were very similar. Displacement experiments were conducted to obtain the apparent dissociation rate constant, koffapp. For this purpose, a TAZ1 mutant was constructed (TAZ1W418Y), where the single tryptophan in the protein was mutated to a tyrosine. A preformed complex solution of TAZ1/TAD-STAT2 (1-2 µM TAZ1 mixed with 1-2 µM TAD-STAT2 variant) was rapidly mixed with varying concentrations of an excess of TAZ1W418Y, in which TAZ1 in complex with TAD-STAT2 is displaced by TAZ1W418Y. The koffapp values were obtained from the single exponential displacement traces since at high TAZ1W418Y concentrations kobs is the same as koffapp. The dissociation constant, Kd, was then obtained as Kd=koffapp/konapp. Double-jump stoppedflow experiments were performed by first mixing TAZ1 with TAD-STAT2 (2.5 µM / 2.5 µM), followed by a controlled delay time, after which displacement by TAZ1W418Y (23 µM) was performed in the second mixing.

8


Page 8 of 28

Page 9 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Calculation of basal association rate constant: The dependence of konapp on ionic strength was fitted to a Debye−Hückel-like approximation (eq. 1)

26

, in order to obtain the basal association

rate constant, kon,basal:

 U  1 ln kon = ln kon ,basal −    RT  1+ κα

(1)

where T is the temperature, R is the gas constant, U is the electrostatic interaction energy, κ is equal to (2NAe2I/ε0εrkBT)1/2, where NA is Avogadro’s constant, e is the elementary charge, I is the ionic strength, ε0 is the vacuum permittivity, εr is the dielectric constant of water, kB is the Boltzmann constant, T is the temperature, and α is the minimal distance of approach and is set to 6 Å26, 27.

Circular dichroism (CD) spectroscopy: Far-uv CD spectra were recorded at 298 K using a Chirascan spectrometer (Applied Photophysics), and a 1 mm cuvette. Protein concentrations were 10 µM TAD-STAT2 and 10 µM TAZ1 in 5 mM HEPES, 50 mM NaCl, 1 mM TCEP (pH=6.9).

Isothermal titration calorimetry (ITC): ITC measurements were performed in 20 mM HEPES (pH=6.9), 190 mM NaCl and 1 mM TCEP, at 293 K, using an iTC200 calorimeter (Malvern Instruments). Proteins were dialyzed against the experimental buffer prior to the ITC measurement. TAZ1 with a concentration of 13.4 µM was placed in the sample cell and 180 µM TAD-STAT2 was loaded onto the syringe. The titration series consisted of a preliminary 0.5 µl injection followed by 20 injections of 1.8 µl. Data were fitted to a one-to-one binding model using software provided by the manufacturer.

9


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 28

RESULTS We have performed a mutational analysis of the coupled binding and folding reaction of TADSTAT2 to TAZ1 in order to characterize the rate-limiting transition state structure for binding. The magnitude of the CD signal and the shape of the CD spectrum of TAD-STAT2 (Figure 2A) are typical for an intrinsically disordered protein, in agreement with the low dispersion in the 15NHSQC NMR spectrum as shown previously23. The CD spectrum of TAZ1 (Figure 2A) displays the characteristics of an α-helical protein, which is in agreement with the NMR structure of free TAZ124.

Figure 2. CD and ITC measurements. A) Circular dichroism spectra at 298 K of 10 µM TADSTAT2 (black) and 10 µM TAZ1 (blue). B) Isothermal titration calorimetry, in which TADSTAT2 (180 µM) was titrated into TAZ1 (13.4 µM).

10


Page 11 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Choice of mutations Transition states are extremely short-lived and they do not accumulate. Therefore, information about the structure of the transition state is obtained indirectly. One way of obtaining such information is by measuring the effects that point mutations have on the binding kinetics (Φvalue analysis, see below). In this work we made 20 mutants in TAD-STAT2 (Table 1). The mutations were made in the interaction surface of TAZ1/TAD-STAT2 based on the previously determined three-dimensional structure of the complex23, and the majority of these mutations were at hydrophobic positions. We also mutated two charged positions that are involved in the formation of solvent exposed intermolecular salt-bridges (R807A and D790A) because we were interested as to what extent these salt bridges affected the binding since complementary electrostatic interactions are prevalent in TAZ1/TAD-STAT2.

Binding kinetics of TAD-STAT2 to TAZ1 The kinetics of association between TAZ1 and TAD-STAT2 were measured at 293 K using the stopped-flow method by varying the concentration of TAD-STAT2 (and its mutant variants) and monitoring the fluorescence change of TAZ1, which contains a single tryptophan (W418). The binding kinetics was biphasic, with a fast and a slow phase (Figure 3A, Figure S1). The kobs for the fast phase increased linearly with TAD-STAT2 concentration with the association rate constant determined to be 5.6 × 106 M-1 s-1, while the kobs for the slow phase was concentration independent with an rate constant value of about 3-4 s-1 at an ionic strength of 0.2 M (Figure 3B).

11


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 28

Figure 3. Binding kinetics of the interaction between TAZ1 and TAD-STAT2. A) A typical stopped-flow binding kinetic trace between TAZ1 (0.75 µM) and TAD-STAT2 (6 µM). Data were fitted to a double exponential function with a fast phase for which kobs depends linearly on TAD-STAT2 (and its mutants) concentration, and a slow phase, with a kobs that is concentration independent with an average value of about 3-4 s-1 for all TAD-STAT2 variants. The inset shows a closer view of the fast phase. B) The observed rate constant, kobs, for the fast and slow (inset) phase as a function of TAD-STAT2 concentration. The concentration dependence of kobs was analyzed using the general equation for association of two molecules25.

12


Page 13 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

The apparent dissociation rate constant, koffapp, determined from displacement experiments, was 1.14 s-1. The Kd determined with the stopped-flow method (0.20 ± 0.01 µM) is in good agreement with the Kd obtained from ITC (0.19 ± 0.02 µM) (Figure 2B). The kobs for the fast phase increased linearly for all TAD-STAT2 mutant variants (Figure S2), The fast phase reports on the ratelimiting transition state for binding. The kobs for the slow phase was concentration independent and were very similar (Figure S2), with an average value of around 3-4 s-1, for all TAD-STAT2 mutants (Table S1, Figure S3). This suggests that this phase is due to a conformational change in TAZ1 that precedes binding. This is also indicated by double-jump experiments in which the first mixing was between TAZ1 and TAD-STAT2, followed by a delay time and with displacement taking place in the second mixing by challenging the reaction mixture with an excess of TAZ1W418Y. The koffapp obtained at a delay time of 0.1 s (koffapp =1.15 s-1) was the same as the koffapp obtained from the regular displacement experiment (Table 1).

Ionic strength dependence The influence of electrostatic interactions on the association kinetics was investigated by studying konapp’s ionic strength dependence26. The konapp is reduced significantly (6.3-fold) by increasing the ionic strength from 0.04 M (konapp = 24.5×106 M-1 s-1) to 1 M (konapp = 3.89×106 M1

s-1), which emphasizes the importance of electrostatic interactions. From the ionic strength

dependence one can also determine the basal association rate constant, kon,basal, which is the rate constant in the absence of electrostatic interactions, using Debye-Hückel theory26. The basal association rate constant for TAZ1/TAD-STAT2 was determined to be about 0.7 ± 0.4 × 106 M-1 s-1 at 293 K (Figure 4).

13


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 28

Figure 4. The ionic strength dependence of konapp for the binding between TAZ1 and TADSTAT2 at 293 K. ln kon values are plotted against (1+κα)-1 and the solid line represents the fit to a Debye-Hückel-like approximation (eq. 1). The correlation coefficient, R, for the linear regression is equal to 0.91. The kon,basal is determined from the intercept with the y-axis, and is 0.7 ± 0.4 × 106 M-1 s-1.

The effect of TAD-STAT2 mutations on the binding to TAZ1 The binding kinetics of 20 TAD-STAT2 mutants were measured at 293 K using stopped-flow fluorimetry (Table 1). Two of the mutations (L795A and F806A) were highly destabilizing for binding and thus did not return reliable kinetics data. Mutations that were made in the Nterminal part of TAD-STAT2 (residues 786-816) clearly have a larger destabilizing effect on the binding to TAZ1 compared to those made at the C-terminal region (residues 817-838) (Table 1, Figure 5B). Indeed, the 3D structure of the protein complex shows that the N-terminal region of TAD-STAT2 is more well-defined and its side-chains are more protruding as compared to the

14


Page 15 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

side-chains in the C-terminal part23. This indicates that the C-terminal part of TAD-STAT2 may retain some flexibility in the bound state, in agreement with the small changes in NMR chemical shifts in this region upon binding23, although a quantitative site-resolved dynamic investigation remains to be carried out. We also mutated two charged positions that are involved in the formation of solvent exposed intermolecular salt-bridges, namely R807 and D790. The side chain of R807 forms a salt bridge with the carboxylate group of E348 in TAZ1 and its backbone carbonyl oxygen makes a hydrogen bond with the side chain amide group of Q355, while D790 forms a salt bridge with R423 in TAZ1. The R807A mutation destabilized the binding by about 0.5 kcal/mol whereas D790A weakened the interaction by only 0.1 kcal/mol. This demonstrates that the net contribution for these interactions to the free energy of binding at an ionic strength of 0.2 M is not substantial.

Table 1. Binding parameters for the interaction between TAZ1 and TAD-STAT2 variants TADSTAT2 mutant WT a D790A L791A b L795A L798A T800S M803A I805A a I805V b F806 R807A V810A I812V I815A a I815V M816A a L822A a A824G a T828S a V829A a V834A

app

kon -1 -1 µM s

app

koff -1 s

Kd µM

∆∆G ∆∆ TS -1 kcal mol

∆∆G ∆∆ Eq -1 kcal mol

Φ

0.20 ± 0.01 0.25 ± 0.02 9.3 ± 1.9

0.08 ± 0.05 0.41 ± 0.11

0.12 ± 0.05 2.23 ± 0.12

 0.18 ± 0.05

5.64 ± 0.26 4.94 ± 0.36 2.80 ± 0.51

1.14 ± 0.03 1.23 ± 0.03 26.0 ± 2.1

8.29 ± 3.35 4.86 ± 0.35 4.64 ± 0.47 4.79 ± 0.56 4.55 ± 0.22

66.6 ± 37.2 2.89 ± 0.07 19.6 ± 0.8 3.46 ± 0.04 1.35 ± 0.04

8.0 ± 5.5 0.60 ± 0.05 4.2 ± 0.5 0.72 ± 0.08 0.30 ± 0.02

-0.22 ± 0.24 0.09 ± 0.05 0.11 ± 0.06 0.10± 0.07 0.12 ± 0.04

2.14 ± 0.40 0.63 ± 0.05 1.77 ± 0.07 0.74 ± 0.07 0.23 ± 0.04

-0.10 ± 0.11 0.14 ± 0.08 0.06 ± 0.04 0.13 ± 0.10 

5.57 ± 0.67 4.95 ± 0.48 4.61 ± 0.34 5.61 ± 0.32 5.68 ± 0.45 4.61 ± 0.22 6.19 ± 0.28 4.50 ± 0.29 5.25 ± 0.39 4.69 ± 0.58 5.31 ± 0.26

2.65 ± 0.26 4.57 ± 0.04 2.90 ± 0.14 1.86 ± 0.08 1.24 ± 0.03 1.93 ± 0.06 1.38 ± 0.04 1.18 ± 0.08 1.16 ± 0.04 1.07 ± 0.02 1.19 ± 0.03

0.48 ± 0.07 0.92 ± 0.09 0.63 ± 0.06 0.33 ± 0.02 0.22 ± 0.02 0.42 ± 0.02 0.22 ± 0.01 0.26 ± 0.03 0.22 ± 0.02 0.23 ± 0.03 0.23 ± 0.01

0.01 ± 0.07 0.08 ± 0.06 0.12 ± 0.05 0.00 ± 0.04 -0.00 ± 0.05 0.12 ± 0.04 -0.05 ± 0.04 0.13 ± 0.05 0.04 ± 0.05 0.11 ± 0.08 0.04 ± 0.04

0.50 ± 0.10 0.88 ± 0.06 0.66 ± 0.06 0.29 ± 0.05 0.05 ± 0.06 0.42 ± 0.04 0.06 ± 0.04 0.15 ± 0.06 0.05 ± 0.06 0.07 ± 0.08 0.06 ± 0.04

0.01 ± 0.15 0.09 ± 0.07 0.18 ± 0.08 0.01 ± 0.15  0.28 ± 0.10     

c

15


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 28

Could not a calculate a reliable Φ-value because of too low ∆∆GEq These mutations destabilized the complex to such an extent that reliable kinetics data could not be obtained. c Determined from fitting to the general equation for the association between two molecules25. Reported errors of konapp represent error from the fitting or the s.d. from repeated measurement determinations of konapp in cases where it was performed, with the largest error reported. Generally these two uncertainties were very similar. The uncertainty for koffapp is the s.d. of kobs at varying concentrations of excess TAZ1W418Y in displacement experiments. a

b

Φ-value analysis and structure of the rate-limiting transition state for binding

In order to obtain a detailed picture of the rate-limiting transition state for binding, a Φ-value analysis was performed26, 28. Φ-binding values were obtained by taking the ratio of the free energy change for the rate-limiting barrier for binding, ∆∆GTS, to the free energy change at equilibrium, ∆∆GEq:

 k wild−type  ∆∆GTS = RT ln  onmu tant   kon   K mutant  d  ∆∆GEq = RT ln  wild−type K  d 

Φ=

∆∆GTS ∆∆GEq

The Kd used in the Φ-value analysis was calculated from the rate constants as Kd=koffapp/konapp. Thus, a Φ-binding value of zero corresponds to a situation where the side chain of the mutated residue does not make any native interactions in the rate-limiting transition state for binding whereas a Φ-binding value of 1 would mean that it makes a full native interaction in the transition state. Values between zero and one are usually interpreted as partial formation of native interactions in the transition state. Here, a Φ-binding value was calculated if the absolute value of

16


Page 17 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

∆∆GEq was equal to or larger than 0.3 kcal/mol. All of the mutations for which we calculated a Φvalue have a ∆∆GEq that is larger than 0.4 kcal/mol, a commonly used cutoff value in Φ-value analysis studies12, 15, 29, except for the I815A mutation with ∆∆GEq=0.3 kcal/mol. This mutation was included in our list of Φ-values, since the precision of the determined rate constants was high. In total, we were able to obtain ten Φ-values that report on the rate-limiting transition-state for binding (Table 1 & Figure 5A).

Figure 5. A) Positions in TAD-STAT2 (pink backbone) are highlighted on the TAZ1/TADSTAT2 structure in yellow for which Φ-binding values could be calculated. All values turned out to be very low (

Native Hydrophobic Binding Interactions at the Transition State for

Recommend Documents