Design of novel granulopoietic proteins by topological rescaffolding.

Birte Hernandez Alvarez,Katherine W Rogers,Kateryna Maksymenko,Laura Weidmann,Masoud Nasri,Andrei N Lupas,Julia Skokowa,Patrick Müller,Karl Welte,Mohammad Elgamacy,Perihan Mir,Murray Coles,Chaitan Khosla

doi:10.1371/journal.pbio.3000919

Abstract

Computational protein design is rapidly becoming more powerful, and improving the accuracy of computational methods would greatly streamline protein engineering by eliminating the need for empirical optimization in the laboratory. In this work, we set out to design novel granulopoietic agents using a rescaffolding strategy with the goal of achieving simpler and more stable proteins. All of the 4 experimentally tested designs were folded, monomeric, and stable, while the 2 determined structures agreed with the design models within less than 2.5 Å. Despite the lack of significant topological or sequence similarity to their natural granulopoietic counterpart, 2 designs bound to the granulocyte colony-stimulating factor (G-CSF) receptor and exhibited potent, but delayed, in vitro proliferative activity in a G-CSF-dependent cell line. Interestingly, the designs also induced proliferation and differentiation of primary human hematopoietic stem cells into mature granulocytes, highlighting the utility of our approach to develop highly active therapeutic leads purely based on computational design.

Highlights

Just as a protein fold can map to different regions of sequence space, a protein function can map to several regions of structure space [1]
The 3D arrangement of the backbone atoms of granulocyte colony-stimulating factor (G-CSF) binding epitope residues in space was disembodied from the native G-CSF structure and used to screen the Protein Data Bank (PDB) for compatible hosting structures, disregarding any sequence similarity to native G-CSF (Fig 1B)
In contrast to the 19 kDa G-CSF, which has the form of an up-up-down-down 4 helix bundle with 2 bundle-interrupting long loops, the 2L4HC2_9 design is a C2-symmetric dimer of a 9 kDa helical hairpin, and the protein from B. halodurans is a 13 kDa up-down 4-helixbundle (Fig 1C). These 2 scaffolds with the G-CSF receptor binding sites modeled in were selected for further optimization in a series of design rounds aiming at hydrophobic corerepacking and hydrophilisation of solvent-exposed residues

Summary

Introduction

Just as a protein fold can map to different regions of sequence space, a protein function can map to several regions of structure space [1]. A molecular function can be potentially achieved through a large number of possible structures, each with a range of possible sequences. While navigating these vast spaces may appear intractable, recent advances in protein design methods have enabled an unprecedented level of control of both sequence and structure [2]. This has paved the way for the rational design of functional proteins to support various applications in synthetic biology [3].

Methods

Results

Conclusion