Prodomain–growth factor swapping in the structure of pro-TGF-β1

Bo Zhao,Chafen Lu,Xianchi Dong,Shutong Xu,Timothy A. Springer

doi:10.1074/jbc.m117.809657

Abstract

TGF-β is synthesized as a proprotein that dimerizes in the endoplasmic reticulum. After processing in the Golgi to cleave the N-terminal prodomain from the C-terminal growth factor (GF) domain in each monomer, pro-TGF-β is secreted and stored in latent complexes. It is unclear which prodomain and GF monomer are linked before proprotein convertase cleavage and how much conformational change occurs following cleavage. We have determined a structure of pro-TGF-β1 with the proprotein convertase cleavage site mutated to mimic the structure of the TGF-β1 proprotein. Structure, mutation, and model building demonstrate that the prodomain arm domain in one monomer is linked to the GF that interacts with the arm domain in the other monomer in the dimeric structure (i.e. the prodomain arm domain and GF domain in each monomer are swapped). Swapping has important implications for the mechanism of biosynthesis in the TGF-β family and is relevant to the mechanism for preferential formation of heterodimers over homodimers for some members of the TGF-β family. Our structure, together with two previous ones, also provides insights into which regions of the prodomain–GF complex are highly structurally conserved and which are perturbed by crystal lattice contacts.

Highlights

TGF-␤ is synthesized as a proprotein that dimerizes in the endoplasmic reticulum
We introduced an R249A mutation into the RHRR249 PC cleavage site between the prodomain and growth factor (GF) domain of human pro-TGF-␤1
We describe here the crystal structure of a pro-TGF-␤1 R249A mutant that is uncleaved by PC and contrast it with a re-refined structure of cleaved pro-TGF-␤1

Summary

Edited by Norma Allewell

TGF-␤ is synthesized as a proprotein that dimerizes in the endoplasmic reticulum. After processing in the Golgi to cleave the N-terminal prodomain from the C-terminal growth factor (GF) domain in each monomer, pro-TGF-␤ is secreted and stored in latent complexes. Swapping has important implications because it can provide a mechanism for preferential formation of heterodimers over homodimers when a cell synthesizes monomers for two different TGF-␤ family members Such heterodimers can display unique biological activities, as in the case of BMP-2/7 heterodimers [20], and can alter activity, as in the case of inhibin heterodimers compared with activin homodimers [12]. We take advantage of two previous structures containing proTGF-␤1 and the current one to provide the first comprehensive analysis of structural differences These structural differences are relevant to TGF-␤ activation because rigidity is important for force transmission, and flexibility is important for release of the GF from the prodomain and for the ability of pro-TGF-␤1 to covalently and noncovalently associate with structurally distinct milieu molecules (6 –9)

Results

No of atoms

The structure around the PC cleavage site

MolProbity percentileg

Evidence for arm domain and growth factor swapping

Missing residues Distance

Discussion

Crystal structures

Protein expression and purification