Mutational Analysis of the Binding-Induced Folding Reaction of the

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Mutational analysis of the binding induced folding reaction of the Mixed Lineage Leukemia protein to the KIX domain Angelo Toto, and Stefano Gianni Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00505 • Publication Date (Web): 24 Jun 2016 Downloaded from http://pubs.acs.org on June 30, 2016

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Biochemistry

Mutational analysis of the binding induced folding reaction of the Mixed Lineage Leukemia protein to the KIX domain Angelo Toto1 and Stefano Gianni 1,2,*

1

Istituto Pasteur Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia

Molecolari del CNR, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”. Sapienza Università di Roma, P.le A. Moro 5, 00185, Rome, Italy. 2

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2

1EW, United Kingdom

*Corresponding author: Stefano Gianni, [email protected]

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ABSTRACT Intrinsically disordered proteins represent a large class of proteins that lack of a welldefined 3D structure in isolation, but can undergo a disorder to order transition upon binding to their physiological ligands. Understanding the mechanism by which these proteins fold upon binding represents a challenge. Here we present a detailed mutational study of the kinetics of the binding reaction between the transactivation domain of the MLL protein, an intrinsically disordered protein, and the KIX domain, performed at different experimental conditions. The experimental data allow to infer the mechanism of folding upon binding and to pinpoint the key interactions present in the transition state. Furthermore, we identify a peculiar malleability of the observed mechanism upon changes of reaction conditions. This finding, which opposes to the robustness typically observed in the folding of globular proteins, is discussed in the context of previous work on intrinsically disordered proteins.


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Biochemistry

The activation of transcription is a key process in the regulation of gene expression and is carried out by diverse proteins that are able to bind DNA and trigger the functions of the RNA polymerase 1. One of the most important proteins involved in these interactions is CBP (cAMP-response element binding protein (CREB)-binding protein), a large multi-domain protein that co-ordinates the function of several transcription factors 2,3. Among others, the KIX domain of CBP is a critical mediator of such interactions

4-7,

being capable to bind the transactivation domains of the different

transcription factors. KIX is a 96-residues globular domain composed by three α-helices (α1-α3) and two 310-helices. Despite its relatively small size, KIX is characterized by the presence of two distinct 8, but energetically connected

5,9-12,

binding sites named

after the their two most characteristic partners, the c-Myb site, named after the transactivation domain of the c-Myb oncoprotein, and the MLL site, named after the mixed-lineage leukemia protein. Importantly, in analogy to other physiological partners of KIX 7, both these domains are intrinsically disordered proteins (IDPs) 13-16, lacking a well-defined three-dimensional structure in isolation and undergoing a disorder-to-order transition upon binding to KIX 4-6,8,12,17,18. MLL is the homologue of Thritorax protein of Drosophila Melanogaster, and it is a member of thritorax protein family

19.

It is involved in the mainteinance of

homeobox (Hox) gene expression during early embryogenesis

20.

We previously

presented a characterization of the binding reaction between KIX and the 19 residues encompassing the transactivation domain of MLL from a kinetic perspective, showing that this process occurs via a co-operative transition, whereby only the disordered unbound and the ordered bound states could be detected, in the absence of any transiently formed intermediates

21.

Notably, by analyzing the pH dependence of the

association (kon) and dissociation (koff) rate constants, we detected a decrease in kon and an increase in koff with decreasing pH, suggesting a scenario in which the rate limiting transition state recognizes the binding pocket of KIX with a partial formation of a salt bridge that is fully formed only in the native state of MLL. Because the observed transitions were consistent with the protonation of a single group of pKa of about 4, we suggested this salt bridge to be either K667(KIX)-D10(MLL) or R624(KIX)-D7(MLL). In this work we present a mutational analysis of the folding upon binding of MLL when binding to KIX; the structure of the complex being reported in Figure 1. By measuring the kinetics of 15 variants, we investigate the structural features of the rate limiting transition state. Furthermore, in the light of our previous characterization of the ACS Paragon Plus Environment

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binding of KIX and MLL

21,

we compare the effect of the mutation of MLL on the

observed reaction at two distinct experimental conditions, i.e. at pH 7.2 and 4.0. A comparison of the data obtained at neutral and low pH allows us to assign the observed pH dependence of kon. to the salt bridge between K667 and D10. Furthermore, the remarkably different effects of the mutations at the two different pH conditions appear to support a scenario whereby the structure of the transition and bound states are malleable with respect to experimental conditions 22. These results are briefly discussed within the frame of previously published work on the binding induced folding of IDPs.

MATERIALS AND METHODS

Site-directed mutagenesis and protein expression and purification

KIX domain, in the form of its Y631W variant, was produced as previously described

21.

The Mixed-Lineage Leukemia protein (MLL) activation domain in its

wild-type form (sequence YNILPSDIMDFVLKNTPSMQALGE) and all its variants were purchased from JPT Peptide Technologies (Berlin, Germany).

Binding experiments

Pseudo-first order binding experiments were performed by using a constant concentration of KIX Y631W of 1-2 µM with increasing concentrations of MLL, typically ranging from 6 to 24 µM. The experiments were performed on an SX-18 stopped-flow instrument (Applied Photophysics, Leatherhead, UK); the excitation wavelength was 280 nm, and the fluorescence emission was measured using a 320 nm cutoff glass filter. The binding experiments were carried out at 10°C, in the presence of 50mM Sodium Phosphate pH7.2, 150mM KCl, or 50mM Sodium Acetate pH 4.0, 150mM KCl. All the experiments were repeated at least three times and the points reported in Figure 2 and 3 are the average of the different experiments. All the measured time courses were consistent with a single exponential. For all the binding experiments the association (kon) and dissociation (koff) rate constant were calculated using the following equation

= [

] +


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Biochemistry

Changes in activation free energy ( ∆∆# ) and equilibrium state free energy (∆∆ ) were calculated as follows

∆∆# =

∆∆ =

RESULTS AND DISCUSSION

The aim of this study is to depict the reaction mechanism of folding upon binding of the transactivation domain of MLL, consisting of a 19 residues IDP, to KIX, a globular domain of 96 residues. A powerful methodology to achieve this goal is based on performing site-directed mutagenesis while measuring the effect of the variations on the association (kon) and dissociation (koff) rate constants

12,22-28.

Thus, we investigated

the binding to KIX of 15 different site-directed mutants of MLL. To measure the reaction, we engineered a construct displaying a fluorescence change upon complex formation, i.e. a KIX variant containing a tryptophan residue in the proximity of the MLL binding site. This variant, namely the pseudo-wild-type Y631W, was also previously used successfully to preliminarily address the reaction 21. We characterized the binding mechanism by mixing a constant concentration of KIX Y631W (1-2 µM) with excess concentrations of the different variants of MLL, typically from 6 to 24 µM. The binding of MLL to KIX is, in theory, a complex reaction involving at least two steps - a recognition event and the folding of MLL. We showed previously that, despite this complexity, the observed kinetics follow a two-state mechanism, without the accumulation of intermediates

21.

In analogy to what observed

for wild-type MLL, all the site directed mutants were consistent with a single exponential time course and displayed a linear pseudo-first order dependence of the observed rate constants, being therefore consistent with a two-state mechanism. Figure 2 summarizes the experimental results obtained for the different variants when


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measured in 50 mM Sodium Phosphate buffer, 150mM KCl at pH 7.2 and 10 °C. The calculated microscopic rate constants for association (kon) and dissociation (koff) are listed in Table 1. Because of the relatively low stability of the KIX-MLL complex, as well as the fast time scale of the observed kinetics, being at the limit of the stopped flow apparatus, several variants, indicated in Table 1, did not return a reliable binding transition and could not be measured. In an effort to observe the binding transition of those variants that were too fast for stopped-flow experiments, temperature jump binding experiments were also tested. Unfortunately however we were not able to detect a measurable binding transition using this technique. A plausible explanation of this observation might be that the binding between KIX and MLL is characterized by a relatively low enthalpy, such that the kinetic amplitudes in T jump experiments are too small to be observed. A quantitative analysis of the binding experiments obtained from a mutational work is typically aimed at calculating the so-called Φ values

29.

In fact, in analogy to

what originally introduced in protein folding studies, by normalizing the effect of a given mutation on the activation versus ground states free energies, it is possible to map interaction patterns present in the rate limiting transition state. The normalization leads to a Φ value, which represents an index of native-like structure content of the transition state, at the level of the mutated residue. However, a critical caveat of the analysis is that mutations should result in a change of binding free energy that should be high enough to be measured reliably, but low enough not to abrogate binding 30. In the case of the variants reported in Table 1, whilst some variants were not possible to measure, was too low (i.e. < 0.4 kcal in the case of some other variants, the value of ∆∆

mol-1) to calculate a reliable value of Φ. Therefore, in the case of the binding induced folding of MLL to KIX, we were not able to produce a complete Φ value analysis, like we previously did on the binding between the transactivation domain of c-Myb and KIX 28.

Nevertheless, as detailed below, the mutational work reported in this paper allows to

draw some interesting conclusions. In a recent study, we analysed the effect of pH on the binding reaction between KIX and MLL

21.

It was found that the logarithm of both association (kon) and

dissociation (koff) rate constants showed a sigmoidal behaviour as a function of pH, compatible with the titration of a single acidic group of pKa of about 4. This finding suggests the partial formation of a salt-bridge in the rate limiting step of the binding reaction. In an effort to test the robustness of the mechanism of binding induced folding ACS Paragon Plus Environment

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of MLL to KIX, we resorted to compare the effect of mutagenesis on MLL at neutral and acidic pH. Thus, the site-directed variants of MLL were measured at pH 4.0, 50 mM Sodium Acetate, 150mM KCl buffer and 10°C. Experimental data obtained at pH 4.0 are reported Figure 3 and the calculated values are listed in Table 1. It is of interest to analyse the structure of the binary complex between KIX and MLL (pdb: 2LXS) on the light of the experimental data reported in Figure 2 and 3. In fact, inspection of the binding pocket reveals the presence of a salt bridge between the MLL residue D10 (highlighted in pink in FIG.1) and the KIX residue K667 (highlighted in light blue). Consistently, in the kinetic data obtained from the mutational analysis reported in Table 1, it may be observed that, while for the D10N mutant kon is decreased by a factor of 2 at pH 7.2 (WT kon = 8.02 ± 0.49 µM-1 sec-1; D10N kon = 4.41 ± 0.68 µM-1 sec-1), the association rate constant in essentially not affected at pH 4.0 (WT kon = 4.21 ± 0.20 µM1

sec-1; D10N kon = 5.45 ± 0.29 µM-1 sec-1). On the contrary, mutation at position D7,

engaging a salt bridge with R624 of KIX, did not show any detectable change in kon as compared to wild-type MLL both at pH 7.2 and 4.0 These finding allows to assign to residue D10 the observed dependence of kon on pH and to conclude the salt bridge between D10 and K667 to be at least partly formed in the rate limiting transition state. In fact, because D10 is partly protonated at acidic pH conditions in the unbound denatured state of MLL, the salt bridge between D10 and K667 is expected to be weaker at low pH rather than at neutral pH. As a consequence of that, the effect of the D10N substitution on kon is more pronounced at pH 7 rather than at pH 4. While the effect of our mutational analysis does not allow drawing a complete Φ value analysis, an interesting observation arises when the quantitative effect of mutagenesis on MLL is compared at high and low pH. Figure 4 is a histogram comparison of the change in activation free energy (∆∆# ) and on the equilibrium state free energy (∆∆ ) of binding for the different variants at pH 4.0 and 7.2. It

is clear that, for nearly all the variants, mutation of a given residue contributes to a different effect on both the activation and ground state free energy, depending on pH. This finding suggests that lowering pH has a pronounced effect on the recognition between KIX and MLL, affecting both the mechanism of binding and the structure of the bound states. In fact, if the structure of the bound state were similar at low and neutral pH, it would be expected to observe a similar change in free energy of binding when mutating MLL at different positions. By analysing the folding upon binding reaction of the transactivation domain of ACS Paragon Plus Environment

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c-Myb to KIX, we recently proposed a general mechanism for the folding of IDPs, named “templated folding”

22.

Following this view, folding upon binding occurs via a

malleable mechanism, which is dictated by the structural features of the binding partner. This type of mechanisms contrasts the robust scenario, typically observed in the folding of globular proteins, where the structure of the transition and native states are largely insensitive to the changes of reaction conditions

30,31.

We speculated the templated

folding mechanism to arise as a consequence of the ability of IDP to achieve specific and reliable binding with multiple partners, while avoiding aberrant interactions. With these premises, we note that the mutational analysis reported in this work appears in agreement with the templated folding mechanism. In fact, whilst binding affinity between KIX and MLL is only marginally affected by changing pH (the KD at neutral and low pH being 10.1 ± 1.1 µM and 29.4 ± 1.7 µM respectively), the mechanism of binding is highly malleable and even the structure of the ground state is affected by the experimental conditions. Future work on different protein systems will be needed to further investigate the generality of the templated folding mechanism for IDP’s.

FUNDING SOURCE INFORMATION

This work was supported by the Italian Ministero dell’Istruzione dell’Università e della Ricerca (Progetto di Interesse ‘Invecchiamento’ to S.G.) and by Sapienza Università di Roma (C26A155S48 to S.G.)

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Table 1. Parameters from kinetic pseudo-first order binding experiments between KIX and MLL variants.

Protein

kon (µM-1 s-1)

WT

8.02 ± 0.49

I3V

koff (s-1) pH 7.2

∆∆G# (kcal mol-1)

∆∆Geq (kcal mol-1)

81 ± 8

-

-

D7N

7.06 ± 0.63 8.71 ± 0.50

82 ± 10 48 ± 8

0.07 ± 0.06 -0.05 ± 0.05

0.08 ± 0.11 -0.34 ± 0.12

I8V

4.80 ± 0.37

49 ± 6

0.29 ± 0.06

0.00 ± 0.10

M9A D10N

5.31 ± 0.50

65 ± 8

0.23 ± 0.06

0.11 ± 0.11

4.40 ± 0.68

138 ± 14

0.34 ± 0.09

0.64 ± 0.12

V12A

3.07 ± 0.13

179 ± 2

0.54 ± 0.04

0.99 ± 0.07

K14A Q20A

6.57 ± 0.75 7.62 ± 0.46

48 ± 15 60 ± 7

0.11 ± 0.07 0.03 ± 0.05

-0.18 ± 0.20 -0.14 ± 0.10

-0.05 ± 0.05

-1.05 ± 0.17

pH 4.0 WT

4.21 ± 0.20

124 ± 4

L4A

4.60 ± 0.31

21 ± 6

D7N

4.24 ± 0.28

103 ± 6

0.00 ± 0.05

-0.11 ± 0.06

I8V

4.59 ± 1.47

77 ± 13

-0.05 ± 0.18

-0.32 ± 0.21

M9A D10N

8.08 ± 0.62 5.45 ± 0.29

20 ± 13 116 ± 5

-0.37 ± 0.05 -0.14 ± 0.04

-1.40 ± 0.36 -0.18 ± 0.05

V12A

4.75 ± 0.25

40 ± 5

-0.07 ± 0.04

-0.70 ± 0.08

K14A Q20A

4.01 ± 0.49

139 ± 8

0.03 ± 0.07

0.09 ± 0.08

4.76 ± 0.38

36 ± 8

-0.07 ± 0.05

-0.77 ± 0.14

The variants L4A, M9G, F11A, F11G, V12G, L13A and L13G at pH 7.2 and I3V, M9G, F11A, F11G, V12G, L13A and L13G at pH 4.0 did not provide reliable binding traces and were excluded from data analysis.


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FIGURE LEGENDS

FIG 1. Three-dimensional structure of the binary complex of MLL (in red) bound to KIX (in green) (pdb: 2LXS). The salt bridge between K667 of KIX (in light blue) and D10 of MLL (in light red) is highlighted in sticks.

FIG 2. Pseudo–first-order kinetics of the binding between KIX held at constant concentration mixed with variable concentration of MLL in wild-type form (black circles fitted by black broken line) and variants (gray squares fitted by gray line) at pH 7.2 and 10 °C.

FIG 3. Pseudo–first-order kinetics of the binding between KIX held at constant concentration mixed with variable concentration of MLL in wild-type form (black circles fitted by black broken line) and variants (gray squares fitted by gray line) at pH 4.0 and 10 °C. (B panel) FIG 4. Effects of point mutations on ∆∆# (A panel) and ∆∆

from Table 1 reported as column graph at pH 4.0 (gray columns) and pH 7.2 (black columns). It is evident that mutagenesis has a different effect at neutral and low pH conditions.


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FIG.1


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FIG.2


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FIG.3


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FIG.4


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Biochemistry

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Biochemistry

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FIG 1. Three-dimensional structure of the binary complex of MLL (in red) bound to KIX (in green) (pdb: 2LXS). The salt bridge between K667 of KIX (in light blue) and D10 of MLL (in light red) is highlighted in sticks. 82x82mm (300 x 300 DPI)


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Biochemistry

FIG 2. Pseudo–first-order kinetics of the binding between KIX held at constant concentration mixed with variable concentration of MLL in wild-type form (black circles fitted by black broken line) and mutants (gray squares fitted by gray line) at pH 7.2 and 10 °C. 81x80mm (300 x 300 DPI)


Biochemistry

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FIG 3. Pseudo–first-order kinetics of the binding between KIX held at constant concentration mixed with variable concentration of MLL in wild-type form (black circles fitted by black broken line) and mutants (gray squares fitted by gray line) at pH 4.0 and 10 °C. 81x79mm (300 x 300 DPI)


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Biochemistry

171x85mm (300 x 300 DPI)


Mutational Analysis of the Binding-Induced Folding Reaction of the

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