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The work described in this chapter focused on identifying which site in dynamin PH domain is involved in phosphoinositide recognition. In addition to site 1 (formed by p- strands pi-p2, p3-p4 and p6-p7) and site-2 (formed by p-strands pl-p2 and p5-p6), a third arginine rich site in dynamin PH domain was identified as a possible candidate Ptdlns(4,5 )? 2 binding pocket. The NMR Ins(l,4,5 )? 3 titration data and the BIAcore

phosphoinositide analysis of the dynamin PH domain mutants presented in this chapter do not implicate either site-2 or the arginine-rich pocket in PtdIns(4,5)P2 binding. Rather, in concurrence with the modelling studies, site 1 was shown to be the phosphoinositide binding pocket of dynamin PH domain.

A detailed three-dimensional representation of the dynamin PH domain binding site can only be achieved by solving its structure in complex with Ins(l,4,5)P3. However, it is possible to build a model of this site together with the predicted PH domain- In s(l,4,5)P3/PtdIns(4,5)P2 interactions from the mutagenesis and NMR solution binding

data (Figure 6.6). In this simplistic model, which does not include ionic and van der W aals interactions between non-lysine residues and In s (l,4,5)P3, the inositol polyphosphate is bound between the loops formed by 1 and 3. Interactions are predominantly hydrogen bonds between the Ins(l,4,5)P3 phosphate groups and the amino side chain of lysine residues. The lysine side chains, Lys 90 and Lys 46/Lys 31 appear to clamp the 4- and 5-phosphate groups of Ins(l,4,5)P3respectively, in the binding pocket. The 1-phosphate group is linked through hydrogen bonds with Lys 27.

The validity of this model is further supported by the negative control mutant, Lys 53. The criteria for selection of this control mutant were that it would be predicted not to be involved in ligand binding, preferably be a lysine residue and lie as proximal to the PtdIns(4,5)P2 binding site as possible. Lys 53 is adjacent to the key phosphoinositide binding residue Lys 54. Mutation of Lys 54 to Met was shown to abolish PtdIns(4,5)P2

binding. Mutation of Lys 53, whose amino side group is diametrically opposed to Lys 54 and so cannot directly participate in ligand recognition, resulted in only a slight loss of PtdIns(4,5)P2 binding.

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Figure 6.6 A model of the dynamin PH domain bound to PtdIns(4,5)P2

A. Representation of the predicted dynamin PH dom ain-PtdIns(4,5)?2 complex. The electro static surface potential is rep resented by a colour range from blue (positive) to red (negative). The positions o f lysine residues involved in specific interactions with PtdIns(4,5)P2 are indicated in black. PtdIns(4,5)P2 is shown with following colour codes: carbon, white; oxygen, red; phophorus, yellow.

B. A three dimensional model of the dynamin PH domain Ins(l,4,5)P3 binding site . H y d ro g e n b o n d s fo rm e d b e tw e en the PH d o m ain ly sin e re s id u e s and Ins(l,4,5)P3 are indicated by dashed lines. The orientation is approxim ately 9(4" to the horizontal axis of A. Ins(l,4,5)P3 is shown with following colour codes: carbon, blue; oxygen, red; phophorus, yellow.

The notion that only the lysines in site 1 of dynamin PH domain mediate binding to Ins(l,4,5)?3 is unlikely given that 2D NMR analysis of the dynamin PH domain also identified several non-lysine residues with significant chemical-shift changes upon the addition of In s(l,4,5)P3. These included Asn 22, Trp 44, Asp 48 and Glu 49. These residues as well as others could play crucial roles in stereo-selectivity and specificity. The three-dimensional structures of PH domain-Ins(l,4,5)P3 complexes have been solved for the p-spectrin (Hyvonen et al., 1995) and PLC 51 (Ferguson et al., 1995) PH domains. These studies demonstrated that the two domains have distinct ligand binding sites. The data for p-spectrin PH domain defines an Ins(l,4,5)P3 binding site formed by loops 1 and

5 (site 1). The contact residues in the spectrin Ins(l,4,5)P3 binding site are Lys 8, Arg 20, Ser 21, Trp 23 and Tyr 70 (Hyvonen et al., 1995). Lys 8, Arg 20 and Trp 23 form part of loop 1, whereas Tyr 70 forms part of loop 5, this pocket being clearly different from that of dynamin PH domain where loops 1, 3 and 6 form the pocket. A binding site similar to dynamin’s is formed by loops 1 and 3 of the PLC 51 PH domain, as shown in the recent X-ray structure (Ferguson et al., 1995). The binding of PtdIns(4,5)P2 to the N-terminal

pleckstrin PH domain has also been analysed by NMR (Harlan et al., 1994). The results show that chemical shift changes, followed as a function of added PtdIns(4,5)P2 were observed for residues located in site-1. These observations emphasise the occurrence of two different potential phosphoinositide/inositol polyphosphate binding sites in PH domains. (Figure 6.7). Site-1 which is utilised by the PH domains of PLC 51 and dynamin PH domains and site-2 by the PH domain of p-spectrin.

The analysis of the PtdIns(3,4,5)P3 binding specificity of the Btk PH domain demonstrated that mutation of Lys 12-Met and Arg 28-Tyr completely abolished this interaction. However, further residues need to be targeted to characterise more precisely the position of the phosphoinositide binding site. The recent determination of the three dimensional structure of a mutant form of the Btk PH domain (Hyvonen and Saraste,

1997) has suggested that although some residues that bind Ins(l,4,5)P3 in the p-spectrin PH domain are conserved in Btk, it is unlikely that the ligand binds in site-2. Moreover, since the Btk PH domain structure shows a clear polarisation of positive charges coincident with the predicted Ins(l,3,4,5)P3 binding site, they proposed that the binding site is similar to that of the PLC 51 and dynamin PH domains i.e. site-1.

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Figure 6.7 A ribbon representation illustrating the two possible binding sites of PH domains

A schematic generalisation of a canonical PH dom ain structure with the variable loop regions 1, 3 and 5 defined as connecting strands p l-p 2 , p3-p4 and P5-P6, respectively. Site-1 formed by loops 1 and 3 is the phosphoinositide binding site for PLC 51 and dynamin, whilst site-2 formed by loops 1 and 5 is the phosphoinositide binding site for the p-spectrin PH domain. Amino- and carboxy- term ini are labelled N and C respectively.