research papers 512 doi:10.1107/S0907444906006962 Acta Cryst. (2006). D62, 512–519 Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 The binding of 2,4-dinitrophenol to wild-type and amyloidogenic transthyretin Eurico Morais-de-Sa ´, a ‡ Ricardo Miguel Neto-Silva, a ‡ Pedro J. B. Pereira, a Maria J. Saraiva a,b and Ana M. Damas a,b * a Instituto de Biologia Molecular e Celular– IBMC, Rua do Campo Alegre 823, 4150-180 Porto, Portugal, and b ICBAS, Universidade do Porto, Largo Prof. Abel Salazar 2, 4099-003 Porto, Portugal ‡ These authors contributed equally to this work and share first authorship. Correspondence e-mail: amdamas@ibmc.up.pt # 2006 International Union of Crystallography Printed in Denmark – all rights reserved Systemic deposition of transthyretin (TTR) amyloid fibrils is always observed in familial amyloidotic polyneuropathy, senile systemic amyloidosis and familial amyloidotic cardio- myopathy patients. Destabilization of the molecule leads to a cascade of events which result in fibril formation. The destabilization of a native protein with consequent conforma- tional changes appears to be a common link in several human amyloid diseases. Intensive research has been directed towards finding small molecules that could work as thera- peutic agents for the prevention/inhibition of amyloid diseases through stabilization of the native fold of the potentially amyloidogenic protein. This work provides insight into the structural determinants of the highly stabilizing effects of 2,4- dinitrophenol on wild-type TTR. It is also shown that similar interactions are established between this molecule and two highly amyloidogenic TTR variants: TTR L55P and TTR Y78F. In the three crystal complexes, 2,4-dinitrophenol occupies the two hormone-binding sites of the TTR tetramer. As a result of 2,4-dinitrophenol binding, the two dimers in the TTR tetramer become closer, increasing the stability of the protein. The three-dimensional structures now determined allow a comprehensive description of key interactions between transthyretin and 2,4-dinitrophenol, a small com- pound that holds promise as a template for the design of a therapeutical drug for amyloid diseases. Received 11 January 2006 Accepted 25 February 2006 PDB References: TTR L55P– DNP, 2b14, r2b14sf; TTR- WT–DNP, 2b15, r2b15sf; TTR T78F–DNP, 2b16, r2b16sf. 1. Introduction Transthyretin (TTR) is a homotetrameric plasma protein that transports thyroid hormones, mainly thyroxine, and also retinol via interaction with the retinol-binding protein (Monaco et al. , 1995; Nilsson et al., 1975). The crystal structure of TTR shows that each TTR monomer consists of a small helical fragment and eight -strands, forming two -sheets (DAGH and CBEF) that assemble as a -sandwich structure (Blake et al. , 1978). The four identical subunits are arranged around a central channel that runs along the protein and in which the binding sites for two thyroxine molecules are located (Wojtczak et al., 1996). Familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis (SSA) are neurodegenerative disorders that result from the aggregation of transthyretin into amyloid fibrils (Saraiva, 2001). The former is a hereditary form of amyloidosis caused by point mutations in TTR, whereas the latter is associated with wild-type TTR (TTR-WT) deposits, predominantly in the heart. Some TTR point mutations result in particularly aggressive forms of FAP and are therefore preferred targets for the investigation of strategies for ther- apeutic intervention. TTR L55P causes the most clinically aggressive FAP variant described so far (Jacobson et al., 1992)