The Carboxyl-terminal Nucleoplasmic Region of MAN1 Exhibits a DNA Binding Winged Helix Domain

Sandrine Caputo,Joël Couprie,Isabelle Duband-Goulet,Emilie Kondé,Feng Lin,Sandrine Braud,Muriel Gondry,Bernard Gilquin,Howard J Worman,Sophie Zinn-Justin

doi:10.1074/jbc.m601980200

Abstract

MAN1 is an integral protein of the inner nuclear membrane that interacts with nuclear lamins and emerin, thus playing a role in nuclear organization. It also binds to chromatin-associated proteins and transcriptional regulators, including the R-Smads, Smad1, Smad2, and Smad3. Mutations in the human gene encoding MAN1 cause sclerosing bone dysplasias, which sometimes have associated skin abnormalities. At the molecular level, these mutations lead to loss of the MAN1-R-Smads interaction, thus perturbing transforming growth factor beta superfamily signaling pathway. As a first step to understanding the physical basis of MAN1 interaction with R-Smads, we here report the structural characterization of the carboxyl-terminal nucleoplasmic region of MAN1, which is responsible for Smad binding. This region exhibits an amino-terminal globular domain adopting a winged helix fold, as found in several Smad-associated sequence-specific DNA binding factors. Consistently, it binds to DNA through the positively charged recognition helix H3 of its winged helix motif. However, it does not show the predicted carboxyl-terminal U2AF homology domain in solution, suggesting that the folding and stability of such a domain in MAN1 depend upon binding to an unidentified partner. Modeling the complex between DNA and the winged helix domain shows that the regions involved in DNA binding are essentially distinct from those reported to be involved in Smad binding. This suggests that MAN1 binds simultaneously to R-Smads and their targeted DNA sequences.

Highlights

The nuclear envelope separates the nucleus from the cytoplasm in eukaryotic cells
Superposition of MAN1CA and MAN1CB spectra shows that all peaks of MAN1CA are found at identical chemical shifts in MAN1CB spectrum (Fig. 1)
This suggests that the three-dimensional structure of MAN1CA is not affected by the presence of the putative UHM domain

Summary

Introduction

The nuclear envelope separates the nucleus from the cytoplasm in eukaryotic cells. It consists of inner and outer nuclear membranes and nuclear pore complexes. MAN1 contains a LEM domain that is present in several proteins, including the inner nuclear membrane proteins lamina-associated polypeptide 2 and emerin [4, 12,13,14] This domain interacts with the DNA and chromatin-binding protein Barrier-to-Autointegration Factor [15, 16]. The entire amino-terminal nucleoplasmic region of MAN1 binds to the nuclear intermediate filaments lamin A and lamin B1 and to emerin [17] It mediates proteinprotein interactions through contacts with the chromatin and the nuclear lamina. We show that the entire carboxyl-terminal region of MAN1 is involved in DNA binding and propose that this interaction is synergetic to the binding of MAN1 to different transcriptional regulators, R-Smads

Results

Discussion

Conclusion