Amide temperature coefficients in characterizing the allosteric effects of ligand binding on local stability in proteins

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Highlights

  • Ligand binding can induce stabilizing effects at distinct regions in a protein.

  • A new method to measure ligand-induced effects on local stability is demonstrated.

  • The method is based on measuring amide temperature coefficients by NMR.

  • Changes in protein stability are observed at regions distant from the interface.

  • The change in stability is proportional to the affinity of the complex.

Abstract

Proteins can stabilize upon binding a ligand. Due to allosteric effects, the changes in stability can occur at regions far from the protein:ligand interface. Efficient methods to measure the changes in local stability upon ligand binding will be useful to understand allostery and may be helpful in protein engineering. In this work, we suggest the measurement of backbone amide temperature coefficients to probe the effect of ligand binding on the local stability of β-sheet rich proteins. The method was applied for two protein:ligand complexes with different binding affinities. The protein includes a beta-sheet network connected by hydrogen bonds. The measured temperature coefficient data captured the stabilizing effect of ligand binding, which propagated across the beta-sheet network of the protein. Intriguingly, the impact on the local and global stability of the protein was proportional to the strength of protein:ligand interaction.

Introduction

Allosteric regulation of protein activity and stability is a central theme in protein biology. Growing evidence suggests that ligand binding can create allosteric changes in a protein to alter its structure, dynamics, and stability [[1], [2], [3]].The effects of ligand binding are often relayed through subtle structural changes from the site of binding to a distant site. The subtle differences are sufficient to modulate local stability in specific regions of the protein. Currently, insights into the ligand-mediated changes in local stability at atomic resolution are inferred indirectly by measuring the motional properties between the free and bound form of the protein. A particular residue in a protein may have a wide range of motions starting from timescales of picoseconds to minutes [4]. Solution state NMR relaxation experiments can provide information about these motions. However, different sets of experiments are required to probe the different ranges of motion, and in some cases, the system is not amenable to the ideal conditions of the experiment. A single efficient technique to measure ligand-induced stability at atomic resolution in proteins would be useful.

Temperature coefficients of backbone amide protons can reflect intramolecular hydrogen bonds in proteins [5,6]. Temperature coefficient values less negative than −4.50 ppb/K indicate intramolecular hydrogen-bonding and more negative than −4.50 ppb/K indicate hydrogen bonding to water molecules [7,8]. The intramolecular backbone hydrogen bonds are a major determinant of stability in an ordered protein. Hence, the 1HNH temperature coefficient can reflect local stability and has been applied to probe differences in local structural stability between wild-type (wt) and mutant proteins [[9], [10], [11], [12]].

In this study, we suggest the use of 1HNH temperature coefficients of backbone amides to characterize allosteric changes in local stability upon ligand binding. The strategy was tested in two protein:peptide complexes with different binding affinities. In both complexes, 1HNH temperature coefficients efficiently detected the allosteric effects of ligand binding on protein stability. A strong correlation was observed between the local as well as global stability of the proteins measured by 1HNHtemperature coefficients and the protein:ligand affinity.

Small Ubiquitin-like Modifier (SUMO) is an important post-translational modifier that regulates multiple biological processes [13]. SUMOylated substrates are recognized by SUMO Interacting Motif (SIM) containing proteins, which then activate downstream processes [14]. One of the many cellular processes regulated by SUMO is host-pathogen interactions [15]. Recently, it was reported that the SIM (SLS4) in the Herpes Simplex Virus protein ICP0 binds to human SUMO to inhibit antiviral responses [16]. Interestingly, upon phosphorylation at two Serine residues in the SIM (ppSLS4), the strength of the SUMO/SLS4 interaction increases [17]. The dissociation constant decreases from 128 μM for SUMO/SLS4 complex to 5 μM for the SUMO/ppSLS4 complex. Atomic Force Microscopy studies have shown that a higher unfolding force is required to unfold SUMO in the SUMO/SIM complex than free SUMO, indicating that SIM binding can stiffen SUMO [18]. Whether the increase in stability of SUMO is solely at the interface or there are other allosteric effects at distance parts of SUMO, is unknown. Here, we investigated the local stability in SUMO1 by 1HNH temperature coefficient experiments using free SUMO1, the SUMO1:SLS4 complex (PDB:6JXU), and the SUMO1:ppSLS4 complex (PDB:6JXV). The results suggest that the SIM SLS4 induces changes in local stability not only at the SUMO1:SLS4 interface, in regions that are distant from the interface. Indeed, 1HNH temperature coefficient experiments can be an efficient tool to probe changes in local stability of β-sheet rich proteins upon ligand binding.

Section snippets

Protein purification

The DNA fragment encoding the 6xHis tagged human SUMO1 in the pQE-80L vector was overexpressed in the E. coli BL21 (DE3). Uniformly 13C/15N-labeled samples were prepared by growing the bacteria in the M9 medium containing 15NH4Cl and 13C6-glucose. For the preparation of uniformly 15N-labeled samples, 13C6-glucose was replaced by unlabelled d-glucose. Cells were grown at 37 °C, and protein expression was induced by the addition of IPTG (isopropyl thio-β-d-thiogalactoside). After 4–5 h of further

Results

We looked into the 1HNH temperature coefficients to assess the changes in the stability of SUMO1 upon binding SLS4. Typically, amide protons in a peptide or a protein show an upfield chemical shift when its temperature is increased. The extent of change in the 1H chemical shift with the change of temperature is unique to its environment and can be directly linked to the local structural stability. The plot of 1HNH chemical shift values with temperature generally shows a linear dependence with

Discussion

The1HNH temperature coefficient measurement could capture the effect of SIM binding on the stability of SUMO. Global average 1HNH temperature coefficients of the SUMO fold confirmed the overall SIM mediated stiffening of SUMO protein. Additionally, the changes in the stability of each secondary structure, and each residue could be individually measured. When compared for SUMO/SIM of different affinities, stronger affinity complexes showed increased stabilization of the native β sheet in SUMO.

Declaration of competing interest

There are no conflicts to declare.

Acknowledgment

The NMR data were acquired at the NCBS-TIFR NMR Facility. R.D. is the recipient of Ramalingaswamy fellowship (BT/HRD/23/02/2006) from the Department of Biotechnology (DBT), Government of India. This research was funded by intramural grants from the National Centre for Biological Sciences, Tata Institute of Fundamental Research.

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