Exploring Protein-Glycan Interactions: Advances in Nuclear Magnetic Resonance

We study protein-protein and protein-glycan interactions, focusing on integrin-disintegrin complex. Our model explores structural dynamics and highlight the role of surface force in mediating complex formation and interaction stability. Our group is dedicated to the study of the role of surface forces in molecular recognition and evolution of binding sites by studying surface hydrophobic clusters present in proteins.

NMR solution enables the detection of protein interactions. Although weak interactions are essential for life, there is a bias towards high-affinity complexes, even though they are present in many important biological functions. Our group is focusing on using integrative structural biology approaches to study large protein complex.

We are using NMR as the main tool to study dynamics and functional aspects. To begin, prepare a four millimolar solution of D-mannose in sodium phosphate buffer at pH 7.4 with 50 millimolar sodium chloride and 5%deuterium oxide. Divide the solution into two samples, one containing 80 micromolar CVN, and the second without.

For mapping the ligand binding through chemical shift perturbation on the NMR spectrometer, set up the HSQCTOPSI pulse sequence for H1C13 HSQC acquisition. Configure the time domain, the spectral width, carrier frequency, and number of scans. Calculate chemical shift perturbation using the equation provided.

To map ligand binding through saturation transfer difference, first, create a new dataset. Configure the parameters for STD NMR experiments by either screening different saturation frequencies or fixing the frequency while varying saturation times to evaluate buildup. Set up 1D H1 NMR experiments using the ZGPR pulse sequence.

Tune the spectrometer for H1, perform shimming, and measure a hard 90 degree pulse. Center the carrier frequency approximately at 4.7 parts per million, corresponding to the water signal, then select the STDDIFFESGP. 3 pulse sequence and set the acquisition parameters.

Set the spectral width as required, interscan delay D1 to four seconds, and define saturation time D20, as per experiment goals. Load the FQ2 list with off-resonance frequency at minus 40 parts per million and on-resonance frequencies to saturate protein only. Test the on-resonance frequencies of minus 0.59, 0.73, and 8.1 parts per million to determine optimal conditions.

Use the optimal frequency to acquire STD spectra at saturation times of 0.5, one, 1.5, two, 2.5, three, and four seconds. Plot the corresponding ASTD values as a function of saturation time. Now, set number of scans to 64 and average the experiment loop four times.

Then, calculate the total number of scans. Set interscan delay to four seconds and acquisition time to 2.7262976 seconds. Configure saturation pulse by setting the duration to 50 milliseconds and control of the Gaussian shape via the shaped program 9SP9.

Apply a T1 row filter in STD NMR variant, then, set the spin lock time D29 based on protein size. To map the ligand binding H1 R2, first, select the CPMG_ESGP2D pulse program from the Bruker standard library. Set the experiment as a 2D acquisition.

Configure the acquisition parameters as shown. Then, run the experiment for 200 cycles of eight scans each. Adjust the variable counter list according to the desired total time of the CPMG cycle.

Run two experiments, one for the sample with the protein and one for the ligand-only sample. Plot the H1 spectra for each TCPMG using the command EFP or sine M followed by FP, then adjust the window function according to the best processing strategy. Calculate the CPMG quotient Q using the equation.

Acquire H1N15HSQC spectra of N15-labeled CVN at 298 Kelvin. Titrate with D-mannose to reach the final concentrations of zero, one, two, five, 10, 20, 40, and 60 millimoles. Use phase-sensitive FHSQCF3GPPH fast HSQC pulse sequence from the Bruker standard library.

Then, calculate the chemical shift perturbation using the formula. Plot CSP values for each residue as a function of D-mannose concentration to determine dissociation constant KD using equation five. Then, fit the resulting data to a single binding isotherm using the formula.

Use phase-sensitive HSQCT2ETF3GPSI3D pulse sequence and configure parameters as shown to measure the N15 R2 values of the individual residues of CVN residues in absence and presence of 60 millimolar D-mannose at 298 Kelvin. Process pseudo-three-dimensional spectra using NMRPipe to generate HSQC-like spectra for each relaxation delay. Import the processed spectra into an analysis platform.

Then, select all spectra and apply the follow intensity changes tool. Plot the intensity of each cross peak as a function of T relax and fit the decay to a monoexponential function to extract N15 R2 values for each residue. Calculate the experimental error from the signal-to-noise ratio of the HSQC-like spectra at 67.84 milliseconds.

Process a region of the spectrum containing only noise and convert it to a text file using the given command. Then, determine the standard deviation of the noise region or noise intensity using statistical software. Compute the experimental error using the given equation.

Chemical shift perturbations were observed for both anomeric forms of D-mannose in the presence of CVN, with greater shifts seen in beta-D-mannose, indicating preferential binding. Two distinct peaks were observed for most D-mannose hydrogens in the presence of CVN, corresponding to free and bound states. The hydrogen nuclei exhibiting the highest ASTD values were associated with beta-D-mannose, consistent with strong bipolar interactions with CVN.

In the transverse relaxation rate analysis, H beta four was the only proton that exhibited a notable increase in relaxation upon binding to CVN. The H1 and N15 chemical shift perturbations induced by the addition of a ligand was highest at concentration up to 10 millimolar. Residues I40, E41, N42, V43, D44, and G45 within the beta strand showed significant chemical shift perturbations, identifying them as the high-affinity binding site for D-mannose.

Binding isotherms showed residue D44 had the highest affinity among the beta strand residues, with a dissociation constant of approximately one millimolar. Measurement of N15 R2 relaxation rates showed both increases and decreases in delta R2 values upon D-mannose binding, suggesting a mix of conformational stabilization and exchange processes. Residue C58, R59, K74, and R76 demonstrated increased delta R2 values, confirming their involvement in the high-affinity binding site, while residues F4, C8, R24, and G27 represented the low-affinity region.