High-Resolution Solid-State NMR Characterization of Ligand Binding

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High-resolution Solid-state NMR Characterization of Ligand Binding to a Protein Immobilized in a Silica Matrix Linda Cerofolini, Stefano Giuntini, Alexandra Louka, Enrico Ravera, Marco Fragai, and Claudio Luchinat J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b05679 • Publication Date (Web): 01 Aug 2017 Downloaded from http://pubs.acs.org on August 2, 2017

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The Journal of Physical Chemistry

High-resolution Solid-state NMR Characterization of Ligand Binding to a Protein Immobilized in a Silica Matrix

Linda Cerofolini,1 Stefano Giuntini,1,2 Alexandra Louka,1,2 Enrico Ravera,1,2* Marco Fragai,1,2,3* Claudio Luchinat1,2*

1. Magnetic Resonance Center (CERM), University of Florence, and Interuniversity Consortium for Magnetic Resonance of Metalloproteins (CIRMMP), Via L. Sacconi 6, 50019 Sesto Fiorentino (FI), Italy 2. Department of Chemistry “Ugo Schiff”, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy 3. GiottoBiotech S.R.L., Via Madonna del Piano 6, 50019 Sesto Fiorentino (FI), Italy

Corresponding Authors: Enrico Ravera: [email protected] Marco Fragai: [email protected] Claudio Luchinat: [email protected]

ACS Paragon Plus Environment

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ABSTRACT Solid state NMR is becoming a powerful tool to detect atomic-level structural features of biomolecules even when they are bound to - or trapped in - solid systems that lack long-range three dimensional order. We here demonstrate that it is possible to probe protein-ligand interactions from a protein-based perspective also when the protein is entrapped in silica, thus translating into biomolecular solid state NMR all the considerations that are usually made to understand the chemical nature of the interaction of a protein with its ligands. This work provides the proof of concept that also immobilized enzymes can be used for protein-based NMR protein-ligand interactions for drug discovery.


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ABBREVIATIONS CP

Cross Polarization

CSP

Chemical Shift Perturbation

DARR

Dipolar Aided Rotary Resonance

DCP

Double Cross-Polarization

hCAII

human carbonic anhydrase II

HEPES

2-[4-(2-hydroxyethyl)piperazin-1yl]ethanesulfonic acid

MAS

Magic Angle Spinning

SSNMR

solid-state Nuclear Magnetic Resonance

swfTPPM

Swept-frequency two-pulse phase modulation

TMOS

tetramethyl orthosilicate


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INTRODUCTION

Several chemical applications including fine chemistry, diagnosis and decontamination rely upon the use of immobilized proteins. The immobilization of enzymes is of particular interest because of the variety of reactions that they can catalyze under mild, physiological, conditions,1–6 reducing the environmental footprint of chemical processing.7 Protein immobilization often results in improved stability;8 furthermore, the immobilized enzyme becomes an heterogeneous catalyst that can be easily removed from the reaction mixture, thus reducing the problems of using enzymes in an industrial setting.9 Several

different

approaches

have

been

proposed

for

the

purpose

of

protein

immobilization,8,10,11 mainly clustered in the categories of: adsorption,12,13 covalent linkage,14–16,6 encapsulation by confinement in a physical barrier17 and entrapment within a matrix.18 Among the entrapment methods, one very interesting approach is the creation of a bioinspired silica scaffold around the protein.19–22 This approach ensures higher immobilization than mere adsorption and, at the same time, does not foresee the creation of any covalent bond, hence it less likely perturbs the native fold of the enzyme. Furthermore, enzymes immobilized through this approach have been reported to have higher stability.8,23,24 Checking the preservation of the active fold of the protein when immobilized often requires a combination of different experimental techniques,25–27 so that most of the characterizations are performed either by the assessment of the enzymatic activity or by immunochemical assays.28 However the recent developments in solid-state biomolecular NMR29–36 have demonstrated that SSNMR is becoming a powerful tool for the characterization of hybrid inorganic/biomolecular composites lacking long range order: evidence has accumulated over the years that it is possible


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to characterize the inorganic component, the composition of the biomolecular component,37–45 changes in the spectra of the entrapped protein46 down to structural features at the atomic level for entrapped47–51 as well as for adsorbed proteins,52 and also to probe the biomolecule-matrix interactions.53–56 Since sufficient resolution is achieved to observe changes in the chemical environment of individual atoms, in principle this would make it possible to probe protein-ligand interactions from the protein perspective. We here describe the application of SSNMR to detect the interaction between silica-immobilized human carbonic anhydrase (hCAII) and two of its inhibitors, the weaker sulpiride and the stronger furosemide. We have chosen hCAII because it has been already characterized by NMR.57–61 hCAII is a very efficient enzyme that catalyzes the hydration of CO2 to HCO3-, which would occur too slowly on the physiological timescale. Immobilized hCA has an industrial relevance for CO2 sequestration,62–66 and its esterase activity67,68 may have biotechnological applications.69 In addition to this, the different isoforms of hCA are found to be overexpressed in several pathologies, thus they are interesting targets for drug development.70–73

MATERIALS AND METHODS Expression of human Carbonic Anhydrase II (hCAII) The gene encoding hCAII into the pCAM vector was transformed into Escherichia coli BL21(DE3) Codon Plus RIPL cells, which were subsequently cultured in rich (LB) medium containing ampicillin (0.1 mg/mL) and chloramphenicol (0.034 mg/mL). Cells were grown at 310 K overnight and then harvested by centrifugation at 4000 rpm (JA-10 Beckman Coulter) for 20 min at 298 K. After switching the cells into the minimal medium containing ampicillin (0,1


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mg/mL) and chloramphenicol (0.034 mg/mL), (15NH4)2SO4 and

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C-glucose, the bacterial cells

were cultured at 310 K for 30 min and subsequently induced with IPTG (1 mM final concentration) and 0.5 mM ZnSO4. Cells were further grown at 310 K for 5 hours and then harvested by centrifugation at 6500 rpm (JA-10 Beckman Coulter) for 20 min at 277 K. All the purification procedures were carried out at 277 K. The pellet was resuspended in 20 mM Trissulfate, pH 8.0, 0.5 mM ZnSO4 (200 mL per liter of culture). Cell disruption was performed on ice by sonication alternating 30 s of sonication and 5 min of resting for 10 cycles. The suspension was ultra-centrifuged at 40000 rpm for 30 min at 277 K. The supernatant, containing crude hCAII protein, was purified by Nickel affinity chromatography using a linear 0-0.5 M Imidazole gradient. Fractions containing pure hCAII were identified by Coomassie staining 13.5% SDS-PAGE gels. Then the protein was dialyzed against 20 mM Tris-sulfate buffer at pH 8.0, 10 mM EDTA and then against 20 mM Tris-sulfate buffer at pH 7.0, 0.5 mM ZnSO4. The hCAII was further purified by gel filtration using a Superdex 75 26/60 column (Amersham Biosciences) in 10 mM HEPES buffer at pH 7.2 and concentrated with Amicon ultrafiltration device to a final concentration of 40 mg/mL. The overall yield of purified protein was 250 mg/L. Silicification reaction The silicification reactions of hCAII were performed as previously reported14 using Poly LLysine as a promoter for the polymerization of silica, but at higher pH (7.2 instead of 7.0) on the other side of the isoelectric point of the enzyme (7.1374). hCAII (400 µL, 40 mg/mL in 10 mM HEPES, pH 7.2), poly-L-lysine (100 µL, 10 mg/mL in H2O; MW: 4000–15000 Da) were added to 1.5 mL vials and shaken for 10 min. To the resulting solution, a 100 mM solution of silicic acid (500 µL) in 10 mM HEPES, pH 7.2 [freshly prepared from a stock solution of 1 M silicic acid obtained by the addition of 1 mM HCl (850 µL) to pure tetramethyl orthosilicate


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(TMOS;150 µL)] was added, and the mixture was left for 10 min at room temperature to react. The suspension was used to fill a 3.2 mm rotor using an ultracentrifugal device.75

NMR measurements All solution NMR experiments for backbone assignment (3D HNCA, 3D HNCO, 3D HN(CA)CO, 3D HNCACB, and 3D CBCA(CO)NH) were performed on a sample of

13

C,15N

hCAII at 0.4 mM protein concentration in 20 mM HEPES, pH 7.5, 0.02% NaN3, with proteases inhibitors, at 310 K, on a Bruker DRX 500 spectrometer equipped with a triple-resonance cryoprobe. All solid-state NMR spectra were acquired with a Bruker AvanceIII HD spectrometer operating at 800 MHz 1H Larmor frequency, equipped with a 3.2 mm E-free HCN probehead, at ~ 291 K; the magic-angle spinning (MAS) frequency was 14 kHz, and 1H decoupling was applied at a nutation frequency of 80 kHz with the swfTPPM scheme.76–79 Samples of hCAII encapsulated in silica matrix were packed into Bruker 3.2 mm zirconia rotors with silicon plugs (courtesy of Bruker Biospin) to preserve hydration.80 90° Pulses were 2.5 µs for 1H, 3.5 µs for 13

C and 5.6 µs for 15N. Interscan delay was set to 2 s. In the 2D 15N-13C NCA experiments, 1H-15N cross polarization (CP)81 was achieved by 1H-15N

CP contact time of 1 ms (ωH = 63 kHz at match, ωN = 35 kHz) with a 100-70 linearly ramped contact pulse on 1H, and the double cross-polarization (DCP)82,83 contact time was 3 ms (ωH = 78 kHz, ωN = 35 kHz, ωC = 21 kHz at match). The number of scans was 1216 for furosemide spectra and 2432 for the initial spectrum of the sample used for interaction with sulpiride. After the loading of the sample with the sulpiride solution the signal decreased and the number of scans was increased to 3600 to partially compensate for the loss in intensity.


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In 2D 13C–13C Dipolar-Aided Rotary Resonance (DARR)84,85 spectrum, the 1H-13C CP contact time was set to 2 ms (ωH = 78 kHz at match, ωC = 50 kHz) with a 100-70 linearly ramped contact pulse on 1H, DARR mixing time was set to 50 ms; the number of scans was 288. All the spectra were processed with the Bruker TopSpin 3.2 software package and analyzed with the program CARA (ETH Zürich).86 The protein backbone assignment was obtained from the analysis of the solution NMR spectra acquired on hCAII and compared with literature data.60,61 The final percentage of assignment was 88%, since residues 1-22 and some residues belonging to loops could not be identified in the spectra, in line with previous reports. 60,61 RESULTS AND DISCUSSION Recently, we have demonstrated that high resolution SSNMR, based on 13C detection, can be applied to observe the spectra of silica-encapsulated enzymes,47 and to monitor the perturbations induced by the silicification with respect to the protein when free in solution.47,48 Incidentally, we note that the silica prepared under these experimental conditions is rather reproducible in terms of quaternary sites distribution,48 and that in previous reports we have not observed any strong site-specific interaction between the protein and the matrix.48,50 We have here applied the same strategy to the characterization of the interaction of hCAII with furosemide, with the aim of proving that it is possible to observe a protein-ligand interaction when the protein is immobilized - entrapped in silica in the present case. The spectra show a remarkable resolution and, at variance with previous observations under different experimental conditions,87,47 protein degradation over time is not observed. Such high resolution is most probably due to the fact that the protein retains its full hydration, and allows for atomic-level


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characterization of the system, whereas in previous investigations by other groups the sample was lyophilized.46 Because of the high resolution achieved in the 2D

15

N-13C NCA spectrum, the assignment

obtained in solution is easily transferable to the solid state. We have thus compared the Cα and N chemical shifts observed in solution with those observed in the solid state for the uninhibited enzyme (Figure 1A). The perturbation is overall minor, with larger differences occurring for some residues on the protein surface, in particular charged residues and residues close to them (Figure 1B), as already observed for other systems.47,48 The preservation of the chemical shift pattern of the active site is already indicative that its structure is preserved, in line with previous enzyme kinetic data on carbonic anhydrase entrapped in silica with a different chemistry87 as well as with the enzymatic activity measured for our composite (see Supporting Information).


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Figure 1. Outcome of the comparison of solution and SSNMR data on hCAII entrapped in the silica matrix: A) chemical shift perturbation (CSP) between free and silica-immobilized hCAII

∆ ∆ according to the formula ∆ = 12 2 + 5 .88 The bars corresponding to the residues exhibiting the largest CSP are highlighted in red. B) CSP mapping on the protein surface (PDB code: 1Z9Y): the charged residues are in blue (positive) and red (negative); the region with the largest perturbation is in magenta.

We also observe that lysine signals in the 2D

15

N-13C NCA show reduced intensities, as

already reported50,89,90 and in line with the expected interaction of positively charged residues with the disordered silica matrix.91–95,54–56 It is interesting to notice that the opposite holds true for the same protein when using an immobilization strategy based on the formation of a covalent linkage of lysine residues with an epoxy-linker grafted on the silica surface.46 This is possibly due to the more homogeneous environment for lysine residues that is achieved by covalent linkage.96,46 The resolution of the solid state NMR spectra is high enough even to allow for effective localization of an inhibitor when bound to the enzyme, as it is done for the so-called proteinbased drug screening in solution.97,98 We have here focused our attention on the detection of the interaction of the furosemide inhibitor with the enzyme. This inhibitor has high affinity (with Kd in the nM range99) for the active site and is thus expected to be in the slow exchange regime with respect to the NMR timescale. Aliquots of inhibitor solutions were added directly in the rotor containing the encapsulated-protein, and the chemical shift perturbations of the protein resonances were analyzed. The new resonances belonging to the inhibited protein could be


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reassigned in the 2D 15N-13C NCA spectrum (Figure 2), also using the information from the 2D 13

C–13C DARR spectrum.

Figure 2. 2D

13

C–15N NCA correlation spectra of uninhibited hCAII (black) and furosemide-

inhibited hCAII (red), encapsulated in the silica matrix using poly-L-lysine. Two of the histidine residues that coordinate the Zn(II), that exhibit the largest chemical shift perturbation, are indicated in the figure.

From the analysis of the chemical shift perturbation (CSP), it is apparent that active site residues exhibit the largest effects (Figure 3A, 3B). The active site of the enzyme (Figure 3C) comprises of three histidine residues that coordinate the Zn(II) (H94, H96, H119); the


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coordination sphere of the metal is completed by a water molecule, which is involved in a hydrogen bonding with T199. This water molecule is removed upon binding to sulfonamide inhibitors (Figure 3D): the NH- moiety of the sulfonamide participates in hydrogen bonding with the Oγ of T199, and one of the oxygen atoms of the –SO2NH- moiety also engages the backbone NH of T199,100–102 explaining the major perturbation at this site. Overall, the residues with the highest CSP on the protein surface are located in the active site cleft, where the inhibitor is bound (Figure 3B,3D).

Figure 3. SSNMR data of hCAII encapsulated in the silica matrix: A) chemical shift perturbation (CSP) between the uninhibited hCAII and hCAII in the presence of furosemide ;88


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the bars corresponding to residues exhibiting the largest CSP are highlighted in red; B) CSP mapping on the protein surface (PDB code: 1Z9Y): the unassigned regions are shown in grey, the region with the largest perturbation in blue, and the inhibitor as yellow sticks; C) focus on the residues exhibiting the largest CPS in the active site, highlighted in blue, on the structure of the free protein (PDB code: 3KS3103) and D) on the protein inhibited with furosemide (PDB code: 1Z9Y); the different coordination spheres of the metal in the two cases are shown.

The method is sensitive enough to reveal differences also in the presence of a lower-affinity inhibitor, sulpiride in the present case. To load the protein with sulpiride, the silica was removed from the rotor, washed with a buffered solution containing sulpiride, previously dissolved in DMSO, and repacked. Also in this case the CSP shows that active site residues exhibit the largest effects (Figure 4A and 4B).


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Figure 4. SSNMR data analysis of hCAII encapsulated in the silica matrix: A) chemical shift perturbation (CSP) between the uninhibited hCAII and hCAII in the presence of sulpiride. The bars corresponding to residues exhibiting the largest CSP are highlighted in red. B) CSP mapping on the protein surface: the unassigned regions are shown in grey, and the region with the largest perturbation in blue. C) CSP mapping on the protein surface after washing of the protein pellet: the unassigned regions are shown in grey, the residues still experiencing a CSP in blue, and the residues with a low intensity signal that disappeared in pink.

CONCLUSIONS AND OUTLOOK In this work, we capitalize on the recent advances in the SSNMR applied to composites having both an inorganic and a biomolecular component. Even if the signal to noise ratio is lower due to the presence of the silicic matrix, the use of high field instruments and dedicated probes mitigates the problem and allows for detection of the protein resonances. We observe a very remarkable resolution, most probably due to the retention of the full hydration of the protein. We here demonstrate that the resolution is high enough to track at the atomic detail the perturbations due to the interaction of the protein with a ligand. This observation allows us to speculate that it will be possible to perform protein-based studies of protein-ligand interactions by means of solid state NMR on immobilized proteins, expanding the possibilities of drug discovery, where immobilized enzymes have already been used for ligand-based screening.28,104–109 A possible pitfall in this strategy is the accessibility of the enzyme by the substrate87 as well as by the candidate drugs, but this is easily checked by performing enzymatic activity tests. We have focused our attention on the selection of the experiments that can be rapidly acquired and at the same time allow for direct translation of the resonances assignments obtained in


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solution for the free target. The present results obtained on the

13

C-15N labelled protein,

combined with the possibility to apply proton detection110,111 to immobilized enzymes,50 suggest that a larger sensitivity and improved throughput will be possible by a proton-detection strategy applied on the 2H-13C-15N labelled protein, exploiting the advantages linked to the larger sensitivity of proton resonances to changes in the local chemical environment. Finally, we expect that by the use of fusion tags to improve immobilization112–115 and of open-ended rotors to be used as “cartridges” for loading with ligand and subsequent washing, it will be possible to test several inhibitors on the same sample.

Supporting Information Experimental details and results of the enzymatic assays on the free and on the immobilized enzyme.

ACKNOWLEDGMENTS This work was supported by Fondazione Cassa di Risparmio di Firenze, MIUR PRIN 2012SK7ASN, the University of Florence CERM-TT and Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI). Specifically, we thank the EU ESFRI Instruct Core Centre

CERM,

Italy.

ER

was

supported

by

FIRC

(grant

number

17941),

and

PhosAgro/UNESCO/IUPAC Green Chemistry for Life.


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High-Resolution Solid-State NMR Characterization of Ligand Binding

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