Atherosclerosis 141 Suppl. 1 (1998) S17 – S24 Molecular modelling and the biosynthesis of apolipoprotein B containing lipoproteins James Scott a, *, Naveenan Navaratnam b , Charles Carter c a National Heart and Lung Institute, Imperial College School of Medicine, Hammersmith Hospital, London W12 ONN, UK b MRC Molecular Medicine Group, Collier Building, Hammersmith Hospital, Du -Cane Road, London W12 ONN, UK c Department of Biochemistry and Biophysics, CB 7260, Uniersity of North Caroline at Chapel Hill, Chapel Hill, NC 27599 -7260, USA Abstract APOBEC-1 is the cytidine deaminase. We show by sequence alignment, molecular modelling and mutagenesis, that it is related in crystal structure to the cytidine deaminase of Escherichia coli (ECCDA). The two enzymes are both homodimers with composite active sites formed with loops from each monomer. In the sequence of APOBEC-1, three gaps compared to ECCDA match the size and contour of the minimal RNA substrate. We propose a model in which the asymmetric binding of one active site to the substrate cytidine which is positioned by the downstream binding of the product uridine and that this helps to target the other active site for deamination. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sequence alignment; Molecular modelling; Mutagenesis 1. Introduction Placental mammals use the two forms of apo B to transport cholesterol and triglyceride in the blood [1]. Full-length apo B-100 (512 kDa) is made in the liver and transports endogenously synthesized cholesterol and triglyceride in the circulation [2,3]. A shorter form, apo B-48 (241 kDa), is generated in intestinal absorp- tive cells by the tissue-specific editing of apo B-100 RNA and is used for dietary lipid absorption [4–7]. The catalytic subunit of the apo B RNA editing enzyme (designated APOBEC-1 for apo B RNA editing cytidine deaminase subunit 1) is a 27 kDa member of the cytidine deaminase family of enzymes that act on monomeric nucleoside and nucleotide substrates [8 – 12]. APOBEC-1 on its own is not sufficient for RNA edit- ing, but acts in concert with other auxiliary proteins The cytidine deaminase family includes the Es - cherichia coli cytidine deaminase (ECCDA). Crystal structures of ECCDA [10] complexed with various in- hibitors [13,14] have been established. ECCDA is a homodimer of identical 31.5 kDa subunits. Each monomer is composed of a small, amino-terminal  - helical domain, and two larger core domains. The two ECCDA core domains have nearly identical tertiary structure, but little apparent amino acid sequence homology. They are connected by an extended chain, running the full length of the molecule from the out- side of the first core domain to the outside of the second. Catalytic activity in ECCDA derives from a cluster of residues in the amino-terminal core domain, which bind zinc and activate a zinc-bound water molecule [10]. The two active sites of the homodimer are formed across the subunit interface. Zinc-binding, catalytic, and pyrimidine-binding sites derive from the amino-ter- minal core domain of one subunit. The rest of the active site comes from a homologous region in the carboxy-terminal core domain of the other subunit, which was described as a ‘pseudoactive site’ because of the structural homology to the active site. * Corresponding author. Tel.: +44-181-3838823; fax: +44-181- 3832028; e-mail: jscott@rpms.ac.uk. 0021-9150/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII:S0021-9150(98)00213-5