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. 2024 Nov 1;80(Pt 11):302-313.
doi: 10.1107/S2053230X24009233. Epub 2024 Oct 14.

Improving the diffraction quality of heat-shock protein 47 crystals

Kevin Kish  1 , Stephen Cobell  1 , Nicolas Szapiel  2 , Chunhong Yan  2 , John A Newitt  2 , Jeffrey Tredup  2 , Iyoncy Rodrigo  2 , Elizabeth Tomasco  2 , Mian Gao  3 , Frank Marsilio  3 , John Haugner  4 , Dasa Lipovšek  4 , Bi Deng  4 , Patrick Bousquet  4 , Yihong Zhang  4 , Holly Schmidt  4 , Steven Sheriff  1
Affiliations

Improving the diffraction quality of heat-shock protein 47 crystals

Kevin Kish et al. Acta Crystallogr F Struct Biol Commun. .

Abstract

Heat-shock protein 47 (HSP47) is a potential target for inhibitors that ameliorate fibrosis by reducing collagen assembly. In an effort to develop a structure-based drug-design system, it was not possible to replicate a previous literature result (PDB entry 4au4) for apo dog HSP47; instead, crystal forms were obtained in which pairs of dog HSP47 molecules interacted through a noncleavable C-terminal His-tag to build up tetramers, all of which had multiple molecules of HSP47 in the asymmetric unit and none of which diffracted as well as the literature precedent. To overcome these difficulties, a two-pronged approach was followed: (i) the His-tag was moved from the C-terminus to the N-terminus and was made cleavable, and (ii) Adnectin (derived from the tenth domain of human fibronectin type III) crystallization chaperones were developed. Both approaches provided well diffracting crystals, but the latter approach yielded crystal forms with only one or two HSP47 complexes per asymmetric unit, which made model building less onerous.

Keywords: C- and N-terminal His-tags; HSP47; crystallization chaperones.

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Figures

Figure 1
Figure 1
Superposition of one tetramer from each of the P21 (PDB entry 9cqe; red), C2221 (PDB entry 9cqf; blue) and P1 (PDB entry 9cqg; cyan) crystal forms, showing that the tetramers superimpose well but the second tetramer in each asymmetric unit is arrayed differently in the three crystal forms. To superimpose PDB entries 9cqe (P21) and 9cqf (C2221) on PDB entry 9cqg (P1), the Cα positions of chains A, B, D and E (a tetramer) were superimposed. The resulting Cα r.m.s.d. was 1.2 Å for 1486 (of 1524 possible) pairs for PDB entry 9cqe on PDB entry 9cqg and 0.8 Å for 1509 (of 1545 possible) pairs for PDB entry 9cqf on PDB entry 9cqg.
Figure 2
Figure 2
Cartoon diagrams of dog HSP47 bound to Adnectins, showing that they bind in the collagen model peptide site. (a) HSP47 (cyan)–Adnectin-44 (magenta) complex (PDB entry 9cqi) showing that a single complex is present in the asymmetric unit. (b) HSP47 (cyan, red)–Adnectin-53 (magenta, blue) complex (PDB entry 9cqj) showing that two complexes are present in the asymmetric unit. (c) Superposition of the HSP47 of the HSP47–Adnectin-53 complex on the HSP47 of the HSP47–Adnectin-44 complex, but showing only the HSP47 (cyan) from the Adnectin-44 complex with Adnectin-44 (magenta) and Adnectin-53 (blue). Although the epitopes of Adnectin-44 and Adnectin-53 overlap, the orientation of the Adnectin relative to HSP47 differs between the two complexes.
Figure 3
Figure 3
Cartoon representation of dog HSP47 showing the five amino-acid differences between the dog and human proteins.
See this image and copyright information in PMC

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