Three-dimensional solution structure of the pleckstrin homology domain from dynamin A. Kristina Downing*, Paul C. Driscoll*t, Ivan Goutt, Kamran Salimt, Marketa J. Zvelebilt and Michael D. Waterfieldt *Oxford University, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, UK. tLudwig Institute for Cancer Research, Courtauld Building, 91 Riding House Street, London WC1 E 8BP, UK. *Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1 E 6BT, UK. Background: The pleckstrin homology (PH) domain is a region of approximately 100 amino acids, defined by sequence similarity, that has been found in about 60 proteins, many of which are involved in signal transduc- tion downstream of cell surface receptors; the function of PH domains is unknown. The only clue to the function of PH domains is the circumstantial evidence that they may link 3 y subunits of G proteins to second messenger systems. Knowledge of the three-dimensional structures of PH domains should help to elucidate the roles they play in the proteins that contain them. Results: Using homonuclear and heteronuclear magnetic resonance spectroscopy, we have determined the solution structure of the PH domain of the GTPase dynamin, one of a number of proteins that have PH domains and interact with GTP. The fold of the dynamin PH domain is composed of two antiparallel 3-sheets, which pack face-to-face at an angle of approximately 600. The first P-sheet comprises four strands (residues 13-58) from the amino-terminal half of the protein sequence; the second P-sheet contains three strands (residues 63-99). A single a-helix (residues 102-116) flanks one edge of the interface between the two sheets, parallel in orientation to the second sheet, in an at/3 roll motif similar to that of the B oligomer of verotoxin-1 from Escherichia coli. Conclusions: The structure of the dynamin PH domain is very similar to the recently reported structures of the pleckstrin and spectrin PH domains. This shows that, despite the low level of sequence similarity between different PH domains, they do have a characteristic polypeptide fold. On the basis of our structure, the sug- gestion that PH domains engage in coiled-coil inter- actions with G protein D3y subunits seems unlikely and should be re-evaluated. Current Biology 1994, 4:884-891 Background Sequence motifs characteristic of a particular type of protein functional domain are often first discovered when two regions within a protein are identified that have related sequences and similar functions. This was true of the pleckstrin homology (PH) domain, which was discovered when similarity was spotted between sequences present at opposite ends of pleckstrin, the major protein kinase C substrate in platelets [1]. Sophisticated sequence-alignment methods subsequently detected putative PH domains in several proteins; many of these proteins have regulatory roles central to the operation of signal-transduction processes in cells [2,3]. Cell growth and differentiation are governed by a network of interactions between proteins involved in signal transduction, cell regulation and gene expression. These proteins frequently have a modular organization, and studies defining the structure and function of their discrete 'modules' have greatly improved our under- standing of their cellular roles. Perhaps the most exciting protein modules defined recently have been the Src homology regions 2 and 3 (SH2 and SH3), which were originally identified by sequence comparisons of Src family transforming proteins and functional analyses of the regions of sequence similarity (reviewed in [4]). SH2 and SH3 regions were subsequently shown to be independently folding domains that bind to specific phosphotyrosine-containing or proline-rich peptide motifs, respectively, thereby mediating protein-protein interactions. Structure determination by nuclear magnetic resonance (NMR) and crystallographic tech- niques, together with binding, mutagenesis and modelling studies, have given us a clear picture of SH2- and SH3-mediated protein-protein interactions [4]. The relative lack of sequence conservation and small size of the PH domain led to the suggestion that it is probably not a catalytic domain, but is more likely to be involved in specific molecular-recognition interactions in a manner analogous to SH2 and SH3 domains [2]. An additional clue to the function of the PH domain has come from its presence in the carboxy-terminal domain of the 3 adrenergic receptor kinase (ARK), where its sequence is almost coincident with a region that has recently been shown to mediate interactions with P 3 y subunits of G proteins [5]. Furthermore, in some systems, the activation of mitogen-activated protein (MAP) kinases by G-protein-coupled receptors is mediated by the D 3 y subunits of G proteins acting on a Ras-dependent pathway [6]; if PH domains are modules for binding G-protein 3y subunits, then this activation process may involve a PH-domain-mediated interaction. Correspondence to: Michael D. Waterfield. © Current Biology 1994, Vol 4 No 10 884