Journal:Acta Cryst F:S2053230X20016015

Using Yeast Surface Display to Engineer a Soluble and Crystallizable Construct of HPK1

Wai L. Lau, Bradley Pearce, Heather Malakian, Iyoncy Rodrigo, Dianlin Xie, Mian Gao, Frank Marsilio, Chiehying Chang, Max Ruzanov, Jodi K. Muckelbauer, John A. Newitt, Dasa Lipovsek and Steven Sheriff ^[1]

Molecular Tour
We were unable to express the wild-type kinase domain (1-346) of hematopoietic progenitor kinase 1 (HPK1, also called MAP4K1). We investigated the potential surface aggregation properties of a model of HPK1 and chose eight sites to investigate by yeast surface display. This led to a double mutant L221D F225E, which expressed in baculovirus-infected insect cells at multiple milligrams per liter of culture. Purification of this construct showed that it was truncated after residue 319, which led to a revised construct of HPK1(1-319) L221D F225E. This construct crystallized in the presence of inhibitors and led to a structure with two molecules/asymmetric unit. The activation loop on both molecules was domain-swapped, that is the activation loop of chain A bind to chain B and vice versa. We were able to use this construct to determine many structures of HPK1/inhibitor complexes in support a structure-based drug design effort.

References

1. ↑ Lau WL, Pearce B, Malakian H, Rodrigo I, Xie D, Gao M, Marsilio F, Chang C, Ruzanov M, Muckelbauer JK, Newitt JA, Lipovsek D, Sheriff S. Using yeast surface display to engineer a soluble and crystallizable construct of hematopoietic progenitor kinase 1 (HPK1). Acta Crystallogr F Struct Biol Commun. 2021 Jan 1;77(Pt 1):22-28. doi:, 10.1107/S2053230X20016015. Epub 2020 Dec 22. PMID:33439152 doi:http://dx.doi.org/10.1107/S2053230X20016015