MBP-binding DARPins facilitate the crystallization of an MBP fusion protein

Rajesh Gumpena; George T Lountos; David S Waugh

doi:10.1107/S2053230X18009901

MBP-binding DARPins facilitate the crystallization of an MBP fusion protein

Acta Crystallogr F Struct Biol Commun. 2018 Sep 1;74(Pt 9):549-557. doi: 10.1107/S2053230X18009901. Epub 2018 Aug 29.

Authors

Rajesh Gumpena¹, George T Lountos², David S Waugh¹

Affiliations

¹ Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
² Macromolecular Crystallography Laboratory, Basic Science Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research Sponsored by the National Cancer Institute, Frederick, MD 21702, USA.

Abstract

The production of high-quality crystals is the main bottleneck in determining the structures of proteins using X-ray crystallography. In addition to being recognized as a very effective solubility-enhancing fusion partner, Escherichia coli maltose-binding protein (MBP) has also been successfully employed as a `fixed-arm' crystallization chaperone in more than 100 cases. Here, it is reported that designed ankyrin-repeat proteins (DARPins) that bind with high affinity to MBP can promote the crystallization of an MBP fusion protein when the fusion protein alone fails to produce diffraction-quality crystals. As a proof of principle, three different co-crystal structures of MBP fused to the catalytic domain of human dual-specificity phosphatase 1 in complex with DARPins are reported.

Keywords: crystallization chaperones; designed ankyrin-repeat proteins; dual-specificity phosphatase 1; maltose-binding protein; surface-entropy-reduction mutagenesis.

open access.

MeSH terms

Amino Acid Sequence
Binding Sites
Cloning, Molecular
Crystallization
Crystallography, X-Ray
Dual Specificity Phosphatase 1 / chemistry*
Dual Specificity Phosphatase 1 / genetics
Dual Specificity Phosphatase 1 / metabolism
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Humans
Maltose-Binding Proteins / chemistry*
Maltose-Binding Proteins / genetics
Maltose-Binding Proteins / metabolism
Models, Molecular
Molecular Chaperones / chemistry*
Molecular Chaperones / genetics
Molecular Chaperones / metabolism
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Interaction Domains and Motifs
Recombinant Fusion Proteins / chemistry*
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism
Sequence Alignment
Sequence Homology, Amino Acid

Substances

Maltose-Binding Proteins
Molecular Chaperones
Recombinant Fusion Proteins
DUSP1 protein, human
Dual Specificity Phosphatase 1

Abstract

MeSH terms

Substances

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