Effect of the Flexible Regions of the Oncoprotein Mouse Double

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Effect of the Flexible Regions of the Oncoprotein Mouse Double Minute X on Inhibitor-Binding Affinity Lingyun Qin, Huili Liu, Rong Chen, Jingjing Zhou, Xiyao Cheng, Yao Chen, Yongqi Huang, and Zhengding Su Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00903 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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Biochemistry

Effect of the Flexible Regions of the Oncoprotein Mouse Double Minute X on Inhibitor-Binding Affinity Lingyun Qin†,, Huili Liu‡, , Rong Chen†,#, Jingjing Zhou†, Xiyao Cheng†, Yao Chen†, Yongqi Huang†,* and Zhengding Su†,* †

Institute of Biomedical and Pharmaceutical Sciences, Key Laboratory of Industrial Fermenta-

tion (Ministry of Education), Hubei University of Technology, Wuhan, 430068, China; ‡

National Center for Magnetic Resonance in Wuhan, State Key Laboratory of Magnetic Res-

onance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Science, Wuhan, 430071, China



These authors equally contributed to this work.

*

Email: [email protected]. *Email: [email protected]

Author Contributions: LQ, RC, JZ and XC carried out the mutagenesis and protein preparation; LQ, RC and HL conducted the NMR analysis; LQ, YC and XC performed the ITC assays; LQ and RC performed the CD and size-exclusion chromatographic analysis; YH carried out the molecular dynamics calculations. ZS, YH and HL designed the experiments and wrote the paper. ZS conceived of the project.

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Abbreviations: MdmX, Mouse double minute X; Mdm2, Mouse double minute 2; HSQC NMR, Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance; p53p, p53 transactivation domain or p53 peptide; NOE, Nuclear Overhauser Effect; ITC, isothermal titration calorimetry; CD, circular dichroism; SEC, size exclusion chromatography; SDS-PAGE, SDS polyacrylamide gel electrophoresis.

TOC:


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Biochemistry

Abstract: The oncoprotein MdmX (Mouse double minute X) is highly homologous to Mdm2 (Mouse double minute 2) regarding their amino acid sequences and three-dimensional conformations, but Mdm2 inhibitors exhibit very weak affinity for MdmX, providing an excellent model for exploring how protein conformation distinguishes and adapts inhibitor-binding. The intrinsic conformation flexibility of proteins plays pivotal roles in determining and predicting the binding properties and the design of inhibitors. Although the molecular dynamics simulation approach enables the understanding of protein-ligand interactions, the mechanism underlying how a flexible binding pocket adapts an inhibitor is less explored experimentally. In this work, we have investigated how the intrinsic flexible regions of the N-terminal domain of MdmX (N-MdmX) affect the affinity of the Mdm2 inhibitor nutlin-3a using protein engineering. Guided by heteronuclear NOEs measurements, we identified the flexible regions that affect inhibitor-binding affinity around the ligand-binding pocket on N-MdmX. A disulfideengineering mutant, N-MdmXC25-110/C76-88, which incorporated two staples to rigidify ligandbinding pocket, allowed high-affinity for nutlin-3a-binding than the wild-type N-MdmX (Kd ~ 0.48 vs. 20.3 µM). Therefore, this mutant provides not only an effective protein model for screening and designing of MdmX inhibitors, but also a valuable clue to enhance the intermolecular interactions of the pharmacophores of ligand with pronounced flexible regions. In addition, our results revealed an allosteric ligand-binding mechanism of N-MdmX that the ligand initially interacts with a compact core, followed by augmenting intermolecular interactions with intrinsic flexible regions. This strategy should be also applicable to many other protein targets to accelerate drug discovery.


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Introduction The binding of overexpressed Mdm2 (Mouse double minute 2) and its homologue MdmX (Mouse double minute X) to the p53 transactivation domain leads to the inactivation of the tumor suppressor p53 and tumor survival in cancer cells, accounting for nearly half of all cancers

(1, 2)

. The inhibition of both Mdm2 and MdmX in cancer cells enable to reactivate

p53 more significantly than agents that only antagonize Mdm2 activity

(3-5)

. Thus, the disrup-

tion of the aberrant interaction of the overexpressed Mdm2 and MdmX with p53 has been treated as an attractive target for anticancer drug discovery (2, 6). The p53 activity in cancer cells is often impaired by the interaction of 15 amino acid sequence within its transactivation domain (referred to hereafter as p53p，Figure 1a). The p53p domain can fold into a helical structure (Figure 1b), when it binds to the N-terminal domains of either Mdm2 or MdmX (referred to hereafter as N-Mdm2 and N-MdmX, respectively, Figure 1c) with the binding affinities of submicromolar concentration

(7)

. The three-

dimensional conformations of N-Mdm2 and N-MdmX are highly similar to each other

(7)

(Figure 1d). The three key residues (F19', W23' and L26') of p53p divide the ligand-binding pockets on N-MdmX and N-Mdm2 into three sub-sites (Figure 1c).


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Biochemistry

Figure 1. Mdm2 inhibitor nutlin-3a exhibits distinctly different affinity for Mdm2 and MdmX. a). The p53p domain is composed of 15 amino acid residues and three key residues (F19', W23' and L26') are depicted in red. b). The p53p folds into a helical structure when binding to either N-MdmX or N-Mdm2. c). N-MdmX is highly homologous to N-Mdm2


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in terms of their amino acid sequences. The secondary structure elements of N-MdmX are illustrated based on the structure of the N-MdmX/p53p complex (3dab.pdb). d). N-MdmX has a similar tertiary structure to NMdm2 when both proteins are in complex with p53p. The three key residues (F19', W23' and L26') from p53p divide the binding pockets into three sub-sites and nutlin-3a mimicking p53p tightly binds to N-Mdm2. F19', W23' and L26', cyan; nutlin-3a, magenta. e). Chemical structure of nutlin-3a, a high-affinity Mdm2 inhibitor.

Many small molecule inhibitors of N-Mdm2 have been developed via different approaches including high-throughput screening and rational designing

(8, 9)

. One of the most

potent Mdm2 inhibitors is nutlin-3a (Figure 1e) with low nanomolar binding affinity (Kd ~ 20 nM) and its analog RG7112 is currently under clinical investigation

(10, 11)

. An X-ray crystal-

lographic structure of the N-Mdm2/nutlin-3a complex indicated that nutlin-3a mimicked p53p peptide to fit very well for the three sub-sites of ligand-binding pocket on Mdm2 (Figure 1d) (10)

. Because N-MdmX is highly homologous to N-Mdm2 in terms of their amino acid se-

quences and three-dimensional conformations, one would take it for granted that N-Mdm2 inhibitors would have similar affinity for N-MdmX. Unfortunately, currently-found Mdm2 inhibitors are very specific for Mdm2 and exhibit very weak affinity for MdmX (6, 8, 12, 13).


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So far, much effort has been made to search effective MdmX inhibitors using Mdm2 inhibitors as scaffolds. In vitro and in vivo investigations on the interaction between MdmX and nutlin analogs suggested that nutlin-3a is a promising scaffold for designing MdmX inhibitors and Mdm2/MdmX dual inhibitors

(14-16)

. Thus, the binding affinity of nutlin-3a ana-

logs has been improved up to around 1 µM via the enhancement of the hydrophobic interaction between nutlin analogs and MdmX (16). One of the promising rational strategies in designing Mdm2 inhibitors was to target the flexible regions of N-Mdm2(17), while targeting the flexible region of N-MdmX for designing MdmX inhibitors remains largely unexplored. Sanchez et al found that the conformation of N-MdmX became relatively rigid in complex with nutlin-3a

(18)

. Grace et al reported that the sub-site of N-MdmX ligand-binding pocket

adjacent to the β-sheets of S1 and S4 was much more flexible than that on N-Mdm2 and they attempted to design few nutlin analogs by targeting the flexible sub-site

(19)

. These investiga-

tions provided feasibility to target flexible regions for assist designing of MdmX inhibitor. However, how individual flexible region and the sub-sites of the ligand-binding pocket on NMdmX affect ligand-binding is still elusive. The intrinsic conformational flexibility of proteins plays pivotal roles in determining and predicting the binding properties and the design of ligands

(20-22)

. Although the molecular

dynamics simulation approach enables the understanding of protein-ligand interactions

(23, 24)

,

the mechanism underlying how a flexible binding pocket adapts a ligand remains largely elusive experimentally. The oncoprotein Mdm2 and MdmX provide excellent examples for exploring how two highly-homologous proteins exhibit distinct differential affinity for ligand in term of protein conformation flexibility. In this study, we attempted to identify the flexible regions on N-MdmX that affect ligand-binding affinity by NMR spectrometry, and then to


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explore the contribution of individual flexible region by protein engineering, aiming to provide a structural rationale for modifying nutlin-3a to augment intermolecular interactions with N-MdmX. Using heteronuclear NOE NMR techniques, we identified four flexible regions on MdmX were correlated to the weak binding affinity of nutlin-3a. Using disulfide staple to rigidify local flexible region, we determined how these regions affected nutlin-3a binding to NMdmX, evaluated by 15N-1H HSQC NMR titration experiments and isothermal titration calorimetry. Finally, a disulfide-engineering mutant that incorporated two staples to rigidify ligand-binding pocket allowed high-affinity for nutlin-3a binding than N-MdmX in 42-folds, providing a more effective protein model for screening and designing of MdmX inhibitors. The protein model also provides valuable clues for specific flexible regions on N-MdmX that should be targeted to augment additional intermolecular interactions for designing highaffinity MdmX inhibitors. Currently the protein model obtained from this work has been utilized to guide for search new fragments to modify nutlin-3a into an MdmX/Mdm2 dual inhibitor, in combination with our recent p53p-N-MdmX fusion protein strategy for highthroughput screening

(25)

. We expect that the methods descripted in this work should be also

applicable to many other protein targets to accelerate drug discovery.


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MATERIALS AND METHODS

Molecular cloning and mutagenesis. The genes encoding the p53-binding domain of human MdmX (N-MdmX, amino acids 22–110) and human Mdm2 (N-Mdm2, amino acids 22–110) were synthesized with E. coli optimized codons and sub-cloned into pET28b plasmid (Novagen, USA) with a Tev protease site and resultant plasmids were defined as pET28-N-MdmX and pET28-N-Mdm2, respectively. The pET28-N-MdmX plasmid was used as a PCR template to construct the expression vector of N-MdmXC25-C110 by replaced the residues at the positions of 25 and 110 into cysteine. Two PCR reactions were performed individually, which one PCR reaction was amplified using T7-forward primer paired with primer-A110C-reverse and another reaction was amplified using primer-N25C paired with T7-reverse primer. The PCR products were directly used as templates for the second PCR amplification, in which only T7 universal primers were used, followed by PCR purification with a PCR purification kit (Qiagen). Subsequently, the purified DNA fragments were treated with restriction enzymes NcoⅠand XhoⅠand subcloned into the pET15b vector, which had been pretreated with the same restriction enzymes. The resultant construct was confirmed with DNA sequencing and defined as pET15b-N-MdmXC25-C110. In N-MdmXC25-C110, the sole intrinsic cysteine at the position of 76 was also substituted with alanine.


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The mutants of N-MdmXC25-C110/C65-C76 and N-MdmXC25-C110/C76-C88 were prepared with the same PCR procedure as above where pET15b-N-MdmXC25-C110 plasmid was used as a PCR template. The protein expression vectors for these mutants were defined as pET15b-NMdmXC25-C110/C65-C76 and pET15b-N-MdmXC25-C110/C76-C88, respectively. To construct a p53p-N-MdmX fusion protein, a DNA fragment containing p53p and a linker was inserted in front of the N-MdmX gene in the pET28-N-MdmX vector. The amino acid sequence of the fusion protein was described previously. Protein expression and purification. All proteins were overexpressed using Escherichia coli BL21 (DE3) strain except disulfide-bonded mutants which were expressed in Escherichia coli Rosetta-Gami B2 strain. Cells were grown in Luria Broth medium, and protein expression was induced with 0.4 mM IPTG at 20C for 12 h except that the expression of disulfide mutants was induced with 0.3 mM IPTG at 25C for 12 h. For 15N-1H HSQC NMR experiments, a protein sample was uniformly labeled with

15

N by growing cells in M9 minimal medium with BME vitamins (Sig-

ma-Aldrich, USA) containing 1 g/L (15NH4)2SO4 as the sole source of nitrogen.

15

N-13C la-

beled NMR samples were prepared using the same medium except that [13C6] glucose (2 g/L) was used as the carbon source. Cells were harvested from 1 L culture by centrifugation at 8000x g for 30 min, resuspended in a lysis buffer, and subjected to periodic sonication for 5–6 min. The lysate was cleared by spinning at 100,000x g, and the supernatant was loaded onto a 20 ml Ni-NTA agarose column (Qiagen). If necessary, the His-tags of the proteins were removed with Tev pro-

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tease digestion at 4C overnight, followed by a second Ni-NTA agarose chromatographic purification. The flow-through was concentrated to 10–12 ml, followed by purification with a S75 gel filtration column. Peak fractions were combined and concentrated to 1 mg/mL. After flash frozen with liquid nitrogen, all protein samples were kept at -80C. An aliquot of each non-isotopically-labeled protein sample was assayed to confirm the formation of disulfide bond by a high-resolutional MALDI Biotyper mass spectrometry (Brucker). Isothermal titration calorimetric (ITC) assay. The protein-ligand interactions were characterized using either an isothermal titration microcalorimeter ITC-200 (Malvern, USA) or Nano-ITC (TA, USA) at 25°C. A typical experiment included the injection of 19 aliquots (2.0 μL each) containing approximately 0.2 mM ligand solution into a protein solution of approximately 10–20 µM in the ITC cell (volume ~ 200 μL). Titrations were performed with stirring speed of 750 rpm and a spacing time of 120 s. A control experiment was run by injecting ligand solution into buffer instead of protein solution in the cell. Before data analysis, the control values were subtracted from the experimental data. The binding isotherms were integrated to give the enthalpy change (ΔH) plotted as a function of the molar ratio of the ligand. When necessary, prior to the integration procedures, the baseline was manually adjusted to minimize the background noise. The initial titration point was always discarded. The Origin 7.0-based software was used for data analysis with the one set of binding site model. The association constant Ka (1/Kd) was determined from the slope of the central linear part of the fractional saturation curve. The Gibbs free energy


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change (ΔG) and the entropy change (ΔS) were calculated based on the following equations: ΔG = −RTlnKa = ΔH − TΔS, where ΔH was derived from the original ΔH/molar ratio plots. NMR experiments. All NMR spectra were collected at 25C on a Bruker Avance 600 MHz spectrometer equipped with a triple-resonance pulse-field gradient probe. 15N-1H HSQC NMR spectra were recorded in the echo-antiecho mode for quadrature detection. All the NMR samples were prepared in a buffer containing 20 mM sodium phosphate, 200 mM NaCl, 0.1% NaN3, and 90% H2O/10% D2O at pH 6.8. DTT (2 mM) was added to the wild-type proteins to prevent the formation of dimers. The final protein concentration was approximately 0.08 mM in free form or 0.4 – 1.0 mM in complex with ligand. All datasets were acquired with 2048 complex points in t2 and 256 complex points in t1. All datasets were processed using TopSpin. Spectral display, assignments, and analysis were performed using the NMRViewJ software package and CARA. Resonance assignment of N-MdmXC25-C110 was obtained manually using

15

N,13C-

labeled sample, using a standard set of triple resonance experiments including HNCA, HNCACB, HNCO and HN(CO)CA spectra. The heteronuclear NOE values were measured at 850 MHz for 15N-labeled protein in complex with nutlin-3a using the pulse sequences described by Farrow et al (26). A 5 s recycle delay was used for the heteronuclear NOE experiment. Steady-state heteronuclear NOE data were obtained in an interleaved manner with and without proton presaturation (3s). Errors for the heteronuclear NOE values were estimated from the root-mean-square variation of noise in empty regions of the two spectra as described previously (27).


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Biochemistry

Circular dichroism (CD) assay. Far UV CD spectra of N-MdmX, N-MdmXC25-C110, N-MdmXC25-C110/C65-C76 and NMdmXC25-C110/C76-C88 were measured in the wavelength range of 190–240 nm with a J-1500 spectropolarimeter (Jasco, Japan) at 25°C in a 1 mm-path-length quartz cell. All CD samples were prepared in a buffer containing 20 mM sodium phosphate, 200 mM NaCl at pH 6.8 except that TCEP (2 mM) was added to N-MdmX sample to prevent dimerization. The buffer solution was used as a control. The CD data was analyzed by Origin 8.0 software. Size exclusion chromatographic assay. Analytical gel filtrations were performed using a GE Health-care SuperdexTM 75 10/300GL column with a flow rate of 0.5 mL/min. 300 µL protein sample at different concentrations was injected. The running buffer contained 20 mM sodium phosphate, 200 mM NaCl at pH 6.8. Data analysis was performed using the ÄKTA™ pure Unicorn software.

RESULTS Effect of Y99 and M53 of N-MdmX on ligand binding. Previously, the configurations of the residues Y99 and M53 on N-MdmX bore the blame for obstructing the entry of Mdm2 inhibitors into the deep binding pocket of N-MdmX (Figure 2a)

(7, 28)

. As such, we were firstly interested in examining the effects of these two

residues on the ligand-binding affinity of p53p and nutlin-3a. After these two residues were mutated into alanine with site-directed mutagenesis, the binding affinities of p53p and nutlin3a for three mutants were determined with the isothermal titration calorimetric (ITC), as listed in Table 1. Figures 2b and 2c show the ITC profiles for the titration of double-mutated N-


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MdmX (N-MdmXM53A/Y99A) with p53p and nutlin-3a, respectively. Although the substitution of these two residues into alanine in either single-mutation (N-MdmXM53A and N-MdmXY99A) or double-mutation (N-MdmXM53A/Y99A) indeed exhibited an improved interaction with p53p, all three mutants exhibited marginal improvement for the interaction with nutlin-3a. These data suggested other factors than the configurations of M53 and Y99 are more dominant in determining the binding affinity of nutlin-3a for N-MdmX, compared with N-Mdm2.

Table 1. The effect of M53A and Y99A mutations on the MdmX-ligand interactions. Kd (µM) N-Mdm2

N-MdmX

N-MdmXM53A

N-MdmXY99A

N-MdmXM53A+Y99A

p53p

0.13  0.02

0.42 0.12

0.51  0.14

0.27  0.11

0.21  0.14

Nutlin-3a

0.02  0.02

20.2  0.32

19.2  0.22

13.2  0.42

> 20


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Biochemistry

Figure 2. The effect of M53A and Y99A mutations on the MdmXligand interactions. a). The configurations of Y99 in N-MdmX (green) and N-Mdm2 (orange) were totally different from each other. The residue M53 from N-MdmX and the residue L53 from N-Mdm2 were shown in stick. Nutlin-3a was depicted in red. b). ITC assays of N-MdmXM53A/Y99A titrated with p53p (left) and nutlin-3a (right), respectively.

Identification of the flexible regions of N-MdmX affecting nutlin-3a-binding. To identify possible conformational factors that could differentiate ligand-binding affinity beyond conformational similarity, we characterized N-MdmX conformation with

15

N-

1

H HSQC NMR spectrometry, in comparison with N-Mdm2. As shown in Figure 3a, the 15N-

1

H HSQC spectrum of the N-MdmX (in black) was superimposed on that of N-Mdm2 (in red)

and the resonance peaks of both spectra had similar dispersion. However, it was notable that the number of resonance peaks was less than expected. Through a series of three-dimensional heteronuclear NMR experiments including 3D_HNCA, 3D_HN(CO)CA and 3D_HNCACB experiments, the appearing resonance peaks in these two 15N-1H HSQC spectra were assignable. For comparison, the resonance peaks of the

15

N-1H HSQC spectra of

15

N-labeled N-

MdmX in complex with p53p and 15N-labeled N-Mdm2 in complex with nutlin-3a were also assigned (Figures S1 and S2).


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Figure 3. Comparison of backbone Conformations between NMdmX and N-Mdm2. a). The assigned

15

N-1H HSQC spectrum of N-

MdmX (black) was superimposed on that of N-Mdm2 (red). Only the residue L81 was labeled for the spectrum of N-Mdm2. b). The

15

N-1H hetero-

nuclear NOE experiments revealed that the backbone flexibility of free NMdmX (top panel) and free N-Mdm2 (bottom panel) were significantly different from each other. The red lines represented average NOE values for individual secondary structure segments that were adapted from the X-ray crystallographic structure.

In the 15N-1H HSQC spectrum of N-MdmX (Figure 3a, black), 27 out of 85 expected resonance peaks were missing because of line-broadening. Those missing signals correspond to the residues I60, M61, V62, Q65, L67, Y68, D69, Q68, Q69, E70, H72, M73, V74, G77, G78, L80, L81, G82, Q88, S89, F90, S91, V92, K93, D94, L107 and V108. In such small protein, their disappearance indicated that some flexible regions on the backbone of N-MdmX undertook intermediate conformational exchange on the µs to ms timescale that is related to the R2 dynamic parameter. On the other hand, the difference in the chemical shift between C and C for each residue along the amino acid sequence of N-MdmX indicated different potential secondary structure segments for those appearing residues (Figure S3a).


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Next we used heteronuclear NOE experiments to evaluate the backbone flexibility of N-MdmX in comparison with N-Mdm2. In general, both proteins had flexible backbone conformation as indicated in Figure 3b. However, N-MdmX had more flexible backbone for the second half of polypeptide chain (the residues 60-111) considering that there were many missing resonance peaks. According to the secondary structure elements of N-Mdm2 in complex with p53p, we averaged the heteronuclear NOE values in segment-by-segment. Thus, it was obvious that five regions on N-MdmX exhibited either similar or different flexibility compared with N-Mdm2. The R-1 region (residues 1-29) and the R-5 region (residues 106-111) have low average NOE values for both N-MdmX and N-Mdm2. The R-3 region (residues 6082) and the R-5 region (residues 88-95) on N-MdmX had much lower average NOE values than those on N-Mdm2, while the R-2 region (residues 40-52) was larger than its counterpart on N-Mdm2. When these regions were mapped on the structure of Mdm2/nutin-3a complex (Figure S3b), they covered all the β-strands and loops, suggesting that the β-strands existed in the protein/ligand complexes formed through a binding and folding process. Figure 4 compares the effects of nutlin-3a binding on the 15N-1H HSQC spectra of NMdmX and N-Mdm2. Titration of nutlin-3a into the 15N-labled N-MdmX in a molar ratio of 2:1 was found to significantly perturb its 15N-1H HSQC resonance peaks (Figure 4a), indicating binding of nutlin-3a specific to certain regions on the surface of N-MdmX. However, many resonance peaks from the residues in the R-3 and R-4 regions were still missing. Such titration was only able to make three new resonance peaks appear from previously-missing peaks (Figure 3a). The three new peaks were from the residues M61, D67 and L81 which were located in the R-3 region. Conversely, these resonance peaks were already detectable in the 15N-1H HSQC spectrum of free N-Mdm2 (Figures 3a and 4b).


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Biochemistry


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Figure 4. Effects of nutlin-3a-binding on the of N-MdmX and N-Mdm2. a). The

15

15

N-1H HSQC spectra

N-1H HSQC spectrum of N-MdmX in

complex with nutlin-3a (red) were superimposed on that of free N-MdmX (black). The three new appeared resonance peaks (M61, D67 and L81) were marked. b). The

15

N-1H HSQC spectrum of N-MdmX in complex with

nutlin-3a (red) were superimposed on that of free N-Mdm2 (black). The resonance peaks for M61, D67 and L81 were marked for both proteins.

Furthermore, we compared the

15

N-1H HSQC spectra of N-MdmX with N-Mdm2

which both were titrated with nutlin-3a (Figure 5a). We found that a unique resonance peak at the cross point of 9.5 ppm in 1H dimension and 123.5 ppm in 15N dimension in the 15N-1H HSQC spectrum of N-Mdm2/nutlin-3a was not present in the

15


MdmX/nutlin-3a. This resonance peak referred to the residue V92 and was observed to be significantly perturbed by the titration of p53p and nutlin analogs to N15-labeled N-MdmX (18)

. The heteronuclear NOEs measurement on N-MdmX/nutlin-3a complexes indicated that

the backbone conformations of N-MdmX around the R-1, R-3, R-4 and R-5 regions were still much flexible even in complex with nutlin-3a, significantly different from N-Mdm2 in complex with nutlin-3a (Figure 5b), given that many resonance peaks were still missing. Nevertheless, it is notable that the heteronuclear NOE values for the observed resonance peaks in NMdmX/ nutlin-3a complexes were significantly enhanced compared with that in free NMdmX (Figure 3b). In agreement, the appearance of L81 resonance peak in the

15

N-1H

HSQC spectrum of N-MdmX suggested that the dynamics of the H3 helix of N-MdmX was


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Biochemistry

changed upon addition of nutlin-3a. Thus, the chemical shift perturbation on the resonance peaks of L81 and V92 could function as indicators for monitoring ligand-binding.


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Figure 5. The R-1, R-3, R-4 and R-regions on N-MdmX still remained flexible compared with N-Mdm2 when both proteins were in the presence of nutlin-3a. a). The

15

N-1H HSQC spectra of N-MdmX

(red) and N-Mdm2 (black) were superimposed when both proteins were in complex with nutlin-3a in the molar ratio of 1:2 (protein : nutlin-3a). The resonance peaks form M61, D67 and L81 were marked for both proteins. The resonance peak of V92 that was only from N-Mdm2 was also labeled. b). When in complex with nutlin-3a, the

15

N-1H heteronuclear NOE data

revealed that the backbone flexibility of N-MdmX in R-3 and R-4 (top panel) were still significantly different from those on N-Mdm2 (bottom panel), while both R-1 and R-5 had similar flexibility. Taken together, although nutlin-3a binding reduced the backbone flexibility of NMdmX, nutlin-3a was still lacking of strong interactions with the R-1, R-3, R-4 and R-5 regions directly or indirectly. This could be one of the major reasons that Mdm2 inhibitors lack high affinity for N-MdmX. Placement of disulfide staples around ligand-binding pocket. As R-1, R-3, R-4 and R-5 were surrounding the ligand-binding pocket on N-MdmX, we were questioning how these flexible regions contributed to the binding affinity of nutlin-3a. To address this question, we engineered disulfide bonds to staple these flexible regions on NMdmX to examine the effect of the flexibility change of local conformation on nutlin-3a binding, as the disulfide bond is a native staple and is easily incorporated into protein structure.


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Biochemistry

Based on the X-ray crystallographic structure of N-MdmX/p53p complex, we selected three pairs of amino acid residues (Asn25/Ala110, Lys65/Cys76 and Cys76/Gln88) on N-MdmX to place the disulfide bond staples, as each pair of these residues had similar distance to a disulfide bond. As shown in Figure 6a, the Cys65/Cys76 disulfide bond was selected to rigidify R-3 region, while the Cys76/Cys88 disulfide bond was selected to rigidify both R-3 and R-4 regions. These two disulfide bonds were located in the rim of the ligand-binding pocket. A third disulfide bond was placed between Cys25/Cys110 to rigidify both R-1 and R-5 regions. Although this disulfide bond was slightly far away from the ligand-binding pocket, both R-1 and R-5 regions on N-MdmX in complex with nutlin analogues exhibited open conformation (Figure S4a)

(19)

, while their counterparts on N-Mdm2 in complex with nutlin-3a were close to each

other (29).


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Figure 6. Rationale for placing disulfide staples to rigidify the ligand-binding pocket of N-MdmX for the enhancement proteinligand interaction. a). An N-MdmX model indicating the locations of three disulfide staples for rigidifying local flexible regions where nutlin-3a was docked into its ligand-binding pocket. b). The

15

N-1H HSQC NMR

spectrum of N-MdmXC25-110 (red) was compared with that of N-MdmX (black) when both samples were in complex with nutlin-3a in the molar ratio of 1:2.

Enhancement of nutlin-3a binding by tethering the R-1 and R-5 regions. We first tethered the R-1 and R-5 regions together with a disulfide staple between Cys25/Cys110. Facilitated with site-directed mutagenesis and a gene-engineered E. coli protein expression system (see MATERIALS AND METHODS), an N-MdmX mutant with a disulfide bond between Cys25 and Cys110 (N-MdmXC25-110) was readily prepared. An aliquot of each non-isotopically-labeled protein sample was assayed to confirm the formation of disulfide bond by high-resolutional mass spectrometry (Figure S5a). By comparing the

15

N-1H HSQC spectrum of N-MdmXC25-110 (Figure 6b, red) with

that of N-MdmX, more peaks were observed when both proteins were incubated with nutlin3a in the molar ratio of 1:2. The

15

N-1H HSQC spectrum of N-MdmXC25-110 was well-

dispersed so that it enabled us to fully assign the resonances through a series of threedimensional heteronuclear NMR experiments. As shown in Figure S4b, almost all expected


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Biochemistry

amide cross peaks of N-MdmXC25-110 in complex with nutlin-3a were detected in the resulting 15

N-1H correlation spectrum. Thus we made sequential backbone assignment of N-MdmXC25-

110

complexed with nutlin-3a using the triple resonance experiments including HNCA,

HN(CO)CA and HNCACB. As results, among 89 amino acids including four proline residues, the resonance peaks of 82 amino acids were assignable (Figure S4b). The three unassignable residues were Q23, Y66 and H72. The residues Y66 and H72 located in R-2 region, while Q23 was in R-1 region (see Figure 1b). Notably chemical shift perturbation was observed for the L81 resonance peak in the R-3 region. We also observed the appearance of the V92 resonance peak in the R-4 region, which was previously characterized as an indicator for monitoring ligand binding (30). It was obvious that tethering the R-1 and R-5 regions was able to affect the interaction of nutlin-3a with the R-3 and R-4 region.

Distinct different effect of the R-3 and R-4 regions on nutlin-3a binding. Subsequently, we put the second disulfide staple to tether the R-4 region on NMdmXC25-110, and generated one double-disulfide bond mutant, named after N-MdmXC25110/C76-88

. The formation of disulfide bonds was confirmed by high-resolutional mass spec-

trometry (Figure S5b). The 15N-1H HSQC spectrum of free N-MdmXC25-110/C76-88 (Figure 7a, black) showed that the two inductor resonance peaks (L81 and V92) were appeared and well dispersed, revealing that the conformations exchange rate of the backbone around L81 and V92 in free N-MdmXC25-110/C76-88 changed into NMR timescale from the µs to ms timescale.


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Figure 7. Addition of second disulfide staple into N-MdmXC25-110 exhibited differential effects on protein-nutlin-3a interaction. a). The

15

N-1H HSQC spectra of N-MdmXC25-110/C76-88 titrated with nutlin-3a in

the molar ratio of 1:0 (black), 1:1 (green) and 1:2 (red). The resonance perturbations for four featured residues (M61, D67, L81 and V92) were zoomed to show titration effect. b). The

15


MdmXC25-110/C76-88 titrated with p53p in the molar ratio of 1:2 (red) was


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Biochemistry

superimposed on that of free N-MdmXC25-C110/C76-88 (black). c). The

15

N-1H

HSQC spectrum of N-MdmXC25-110/C65-76 (black) was superimposed on that of N-MdmX (red). d). The

15

N-1H HSQC spectra of N-MdmXC25-110/C65-76 ti-

trated with nutlin-3a in the molar ratio of 1:0 (black), 1:0.5 (yellow), 1:1 (green) and 1:2 (red) were shown with partial resonance assignments. The resonance perturbations for some residues were zoomed to show the titration effect.

We also observed that the

15

N-1H HSQC resonance peaks of N-MdmXC25-110/C76-88

were significantly perturbed upon nutlin-3a titration (Figure 7a), indicating a relativelystrong binding of nutlin-3a to N-MdmXC25-110/C76-88 more than to N-MdmXC25-110 and NMdmX. On the other hand, this double disulfide-bonded mutant still maintained a bone fide interaction with p53p, as examined with a ure 7b and Figure S6), where the

15

15

N-1H HSQC NMR titration measurements (Fig-

N-1H HSQC resonance peaks were significantly per-

turbed. Next, we introduced a disulfide staple between Cys65 and Cys76 to tether the R-3 region on N-MdmXC25-110 and generated another double disulfide-bonded mutant (N-MdmXC25110/C65-76

). Figure 7c showed that resonance peaks present in the 15N-1H HSQC spectrum of N-

MdmXC25-110/C65-76 were less than in that of N-MdmX. Nevertheless, these appearing resonance peaks were still dispersed, suggesting that the some more backbone N-MdmXC25-110/C6576

fell into intermediate conformation than the wild-type N-MdmX on the µs to ms timescale.

Through heteronuclear three-dimensional NMR experiments, we were able to partially assign


Biochemistry

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these resonances and found that those missing NMR signals mainly came from the R-4 region and the helix H4 (Figure 7d). Nevertheless, these dispersed resonance peaks could be significantly perturbed upon titration of nutlin-3a, indicating that a binding process occurred. However, the binding of nutlin-3a was not able to make missing peaks reappear, which happened to the wild-type N-MdmX.

Disulfide staples did not alter the secondary structures of N-MdmX. To ensure that the introduction of disulfide bones would not affect the global structure of N-MdmX, we conducted circular dichroism (CD) spectroscopic assay on the three mutants in far-UV range from 190 nm to 240 nm. As shown in Figure 8a, CD spectra revealed that all the disulfide-bonded mutants still maintained similar secondary structures to those of NMdmX, indicating that introducing these disulfide staples did not change the overall secondary structures of N-MdmX.


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Biochemistry

Figure 8. Disulfide staples did not alter the secondary structures of N-MdmX. a). Comparison of the CD spectra of the three disulfide-bonded mutants with that of N-MdmX. b). The NMR samples were analyzed using SEC and SDS-PAGE, compared with a fusion protein of p53p-N-MdmX.


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To exclude the possibility that the NMR signal disappearance happened to NMdmXC25-110/C65-76 could be caused by protein aggregation, we further characterized the conformational properties of these mutants using size exclusion chromatography (SEC) and nonreduced SDS-PAGE analysis. All NMR samples were directly applied to SEC while a single chain polypeptide of p53p-N-MdmX fusion protein was used as a control

(25)

. As shown in

Figure 8b, all SEC profiles appeared as symmetric peaks, indicating no aggregation happened in the NMR samples. Notably the hydrodynamics of the mutant N-MdmXC25-110/C65-76 indicated conformational population, which could mean conformational population exchange between open conformation and closed conformation for the ligand-binding pocket. Nevertheless, our mass spectrometric assay suggested that no free cysteine existed for N-MdmXC25110/C65-76

. Furthermore, the non-reduced SDS-PAGE analysis indicated that no intermolecular

cross-linking happened (Figure 8b, inset). Quantitative determination of the binding affinity of nutlin-3a to disulfide-bonded mutants. Furthermore, we used ITC to quantitate the binding affinity of nutlin-3a for these mutants. Except that ITC could not detect enthalpy change for the titration of nutlin-3a to NMdmXC25-110/C66-76 (data not shown), ITC assays confirmed that nutlin-3a exhibited higher binding-affinity for both N-MdmXC25-110/C76-88 and N-MdmXC25-110 than N-MdmX. As shown in Figures 9a and 9b for N-MdmXC25-110/C76-88 and N-MdmXC25-110, respectively, their ITC thermodynamic profiles exhibited good transition as a function of the molar ratio of protein to nutlin-3a, compared with N-MdmX titrated with nutlin-3a (Figure S7). On the other hand, both p53p exhibited better affinity to N-MdmXC25-110/C76-88 than to N-MdmX (Figures 9c),


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Biochemistry

while p53 still maintained medium affinity for N-MdmXC25-110 (Figures 9d) similar to NMdmX (Figure S7).


Biochemistry

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Figure 9. Thermodynamic characterization of two significant disulfide-bonded mutants of N-MdmX titrated with nutlin-3a or p53p. a & b). ITC assays of N-MdmXC25-110/

C76-88

titrated by nutlin-3a and p53p,

respectively. c & d). ITC assays of N-MdmXC25-110 titrated by nutlin-3a and p53p, respectively.

As summarized in Table 2, rigidifying different flexible regions significantly affected the binding affinity of nutlin-3a. Compared with N-MdmX, nutlin-3a exhibited 10-fold enhanced binding-affinity for N-MdmXC25-110. After adding the Cys76/Cys88 disulfide bond into this mutant, nutlin-3a exhibited a dramatically enhanced binding for this double disulfidebonded mutant N-MdmXC25-110/C76-88 with a Kd value of 0.48 µM. On the contrary, after adding the Cys65/Cys76 disulfide bond into N-MdmXC25-110, nutlin-3a exhibited a dramaticallyreduced binding affinity for this double disulfide-bonded mutant N-MdmXC25-110/C65-76 to the extent which was not detectable with ITC (data not shown). On the other hand, rigidifying different flexible regions also significantly affected the binding affinity of p53p following a similar trend to that observed for nutlin-3a (Table 2). The p53p exhibited a comparable binding affinity for N-MdmXC25-110 (Kd = 0.71 µM) and N-MdmX, but an enhanced binding affinity for N-MdmXC25-110/C76-88 (Kd = 0.02 µM) in 21-fold stronger than the binding affinity of p53p for N-MdmX. However, unlike nutlin-3a, p53p also exhibited a marginally-reduced binding affinity N-MdmXC25-110/C65-76.


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Biochemistry


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DISCUSSION and CONCLUSION Previously it was reported that the binding of nutlin-3a could rigidify the N-MdmX conformation (18, 19), although this Mdm2 inhibitor exhibited a marginal binding affinity for NMdmX. In this work, we in detail investigated the effect of the flexible regions around the ligand-binding pocket of N-MdmX on ligand-binding affinity, using disulfide staple in combination with NMR spectrometry and ITC. We found that four flexible regions (R-1, R-3, R-4 and R-5 in Figure 6a and Figure S3b) that played different roles in determining the binding affinity of nutlin-3a. After tethering the R-1 and R-5 regions, the appearance of many new resonance peaks in the 15N-1H HSQC spectrum of N-MdmXC25-C110 titrated by nutlin-3a occurred (Figure 6c) in company with the enhanced binding affinity of nutlin-3a (Table 2), suggesting that the interaction of nutlin-3a with R-3 and R-4 regions could be allosterically enhanced in the wildtype N-MdmX. Further constraining R-4 region by introducing the Cys76/Cys88 disulfide bond resulted in a preformed bound-like ligand-binding pocket on N-MdmXC25-110/C76-88 as revealed from the resonance perturbation in its 15N-1H HSQC spectrum (Figures 7a and 7b) and the enhanced binding affinity of nutlin-3a (Table 2). On the contrary, introducing the Cys65/Cys76 disulfide bond deformed the R-3 region and rendered a partial folded ligandbinding pocket on N-MdmXC25-110/C65-76, as indicated by its 15N-1H HSQC spectrum (Figures 7c and 7d). Obviously the

15

N-1H HSQC titration of N-MdmXC25-110/C65-76 with nutlin-3a

(Figure 7d) indicated that this partial folded conformation was still able to interact with nutlin-3a. Nevertheless, our CD analysis indicated that N-MdmXC25-110/C65-76 did not lose its secondary structure, compared with N-MdmX (Figure 8a).


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Biochemistry

Our thermodynamic data suggested a clear direction how to modify nutlin-3a into an MdmX inhibitor. As revealed from Table 2, rigidifying R-1 and R-5 significantly contributed to protein-ligand interaction by increasing enthalpy (0.33 kcal/mol) and entropy (1.08 kcal/mol) as well. Further rigidifying R-4 region on the basis of rigidified R-1 and R-5 regions enhanced protein-ligand interaction by increasing enthalpy (0.42 kcal/mol) and entropy (0.39 kcal/mol). To achieve a similar high-binding affinity of nutlin-3a for N-MdmX existed for N-Mdm2, an ideal nutlin analog of MdmX inhibitor should not only enable to rigidify R1, R4 and R5 regions in entropy manner, but also need additional 2.26 kcal/mol of interaction in enthalpy manner. This rationale was also observed from the thermodynamic parameters of p53p interacting with N-MdmX and its disulfide mutants. Rigidifying R1, R4 and R5 regions made protein conformation more favorable for p53p-binding, achieving 10-fold stronger affinity. Therefore, as summarized in Figure 10, the interaction of nutlin-3a with N-MdmX was highly correlated with the conformational rigidity. Among four flexible regions around the ligand-binding pocket on N-MdmX, different regions were responsible for the ligand recognition and stabilization. On the other hand, as protein plasticity manifests itself first and foremost in the kinetics of ligand binding, rapid kinetics data that can be obtained from stoppedflow experiments should be supportive and informative for understanding the effects of these flexible regions on ligand-binding pathway.


Biochemistry

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Figure 10. Summary of the effects of conformational rigidity of NMdmX on nutlin-3a binding. The conformations of N-MdmX and its three disulfide mutants were generated based on the snapshots in the MD simulation (see Figure S8 in Supporting Information) based on an X-ray crystal structure of N-MdmX in complex with p53p.


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Biochemistry

Although a detailed structural investigation is required to understand the molecular mechanism of ligand-binding for these disulfide-bonded N-MdmX proteins, our current data suggested that the interaction of a weak ligand with the oncoprotein MdmX can be enhanced by tuning the intrinsic flexibility of its ligand-binding pocket. In practice, it is not reasonable to generate a disulfide-bonded MdmX mutant in cancers, but our results provide reversethinking strategy how to start to modify nutlin-like ligands for increasing the intermolecular interaction between nutlin analogs and the wild-type MdmX. More specifically, new MdmX inhibitors should be focused on improving the interactions between pharmacophores and the R-4 regions in addition to the interaction with R-1 and R-5 regions simultaneously. In a similar strategy, Bista et al had successfully used the flexible region of Mdm2 in designing the inhibitors of this closely-related protein-protein interaction previously (17). Importantly, our work suggested that the disulfide staples could be used as a molecular tool to identify hotspots on N-MdmX for building up new intermolecular interactions with nutlin analogs. Therefore, we are currently using this strategy for guiding the modification of nutlin-3a into an MdmX/Mdm2 dual inhibitor. In combination with our recent p53p-N-MdmX fusion protein strategy for high-throughput screening

(25)

, we have found that benzodiazepin

can fit into F19-site on N-MdmX for rigidifying the R-3 region (unpublished data). Therefore, it is expected that a potent MdmX inhibitor should have the ability to interact with the R-3 and R-4 regions additively and allosterically. Such potent inhibitor should also exhibit a high affinity to Mdm2, as predicted previously

(6)

. More significantly, the reverse-engineering

strategy described in this work provides a novel strategy for designing high-affinity MdmX inhibitors and dual MdmX/Mdm2 inhibitor. This strategy should be also applicable to many other protein targets to accelerate drug discovery.


Biochemistry

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Acknowledgements This research was supported by National Natural Science Foundation of China (YQH, 21603121) and (YC and ZDS, 31500132), State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics (ZDS and HLL, T152604), Hubei Provincial Innovative Project (ZDS, 2016ACA128) and Wuhan Natural Science Foundation (ZDS, 201506101010033).

Competing financial interests The authors declare no competing financial interests.

Supporting Information Available Supplementary Results and Discussions are available in the online version of this paper.


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