Structure-based design approach to rational site-directed mutagenesis of β-lactoglobulin

Piotr Bonarek,Joanna I Loch,Magdalena Tworzydło,David R Cooper,Katažyna Milto,Paulina Wróbel,Katarzyna Kurpiewska,Krzysztof Lewiński

doi:10.1016/j.jsb.2020.107493

Piotr Bonarek, Joanna I Loch + Show 6 more

Open Access

https://doi.org/10.1016/j.jsb.2020.107493

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Abstract

Recombinant proteins play an important role in medicine and have diverse applications in industrial biotechnology. Lactoglobulin has shown great potential for use in targeted drug delivery and body fluid detoxification because of its ability to bind a variety of molecules. In order to modify the biophysical properties of β-lactoglobulin, a series of single-site mutations were designed using a structure-based approach. A 3-dimensional structure alignment of homologous molecules led to the design of nine β-lactoglobulin variants with mutations introduced in the binding pocket region. Seven stable and correctly folded variants (L39Y, I56F, L58F, V92F, V92Y, F105L, M107L) were thoroughly characterized by fluorescence, circular dichroism, isothermal titration calorimetry, size-exclusion chromatography, and X-ray structural investigations. The effects of the amino acid substitutions were observed as slight rearrangements of the binding pocket geometry, but they also significantly influenced the global properties of the protein. Most of the mutations increased the thermal/chemical stability without altering the dimerization constant or pH-dependent conformational behavior. The crystal structures reveal that the I56F and F105L mutations reduced the depth of the binding pocket, which is advantageous since it can reduce the affinity to endogenous fatty acids. The F105L mutant created a unique binding mode for a fatty acid, supporting the idea that lactoglobulin can be altered to bind unique molecules. Selected variants possessing a unique combination of their individual properties can be used for further, more advanced mutagenesis, and the presented results support further research using β-lactoglobulin as a therapeutic delivery agent or a blood detoxifying molecule.

Highlights

Recombinant proteins are a growing group of innovative therapeutic agents with applications in various medical disciplines
L39 located on the AB loop occupies a position at the binding pocket entrance and its side chain fills the space between loops AB and GH, close to EF and GH loops involved in Tanford transition (Sakurai and Goto, 2006)
To decide which amino acids are the most promising alternatives to the natural residues, we considered several of their properties, namely hydrophobicity, ability to form specific interactions with ligands, potential influence on the binding pocket geometry, and the potential stabilizing effect of substitution resulting from the van der Walls interactions to neighboring residues

Summary

Introduction

Recombinant proteins are a growing group of innovative therapeutic agents with applications in various medical disciplines. This group of biopharmaceuticals is diverse and includes hormones, cytokines, growth factors, plasma proteins, enzymes, inhibitors, coagulation factors, fusion proteins, antigen-binding fragments (Fab), antibodydrug conjugates, and monoclonal antibodies (Lagassé et al, 2017; Shepard et al, 2017). Anticalins have many advantages over classical therapeutic antibodies; they are relatively small with a compact β-barrel fold and have a high structural stability, high target specificity, low immunogenicity, and low cost of production in E. coli cells (Gebauer and Skerra, 2012; Richter et al, 2014). Mutations are introduced to the region of flexible loops allowing the substituted residues to interact with molecular targets such as receptors (Richter and Skerra, 2017) (Anderson et al, 2015), peptides (Gille et al, 2016; Hohlbaum et al, 2018), or small molecules (Barkovskiy et al, 2019; Dauner and Skerra, 2019; Eggenstein et al, 2014)

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