Efectos sobre la financiación 1 Resultados de estudios cuantitativos

Biochemical studies revealed that VHL forms a ternary complex with the Elongin C and Elongin B proteins [98, 108]. The VHL-Elongin C-Elongin B complex (henceforth VCB complex) has a central role in VHL function because most of the tumour-derived mutations destabilise this complex [98, 108, 109].Furthermore, peptide mapping studies showed that a 12-amino acidregion of VHL that contains nearly a quarter of the tumour- derivedmutations makes key interactions with Elongin C [109],tightly linking the Elongin C binding and tumour suppression functions of VHL. The following section outlines the biochemical structural analysis of a pVHL–Elongin C and pVHL-HIF1α interaction.

2.9.1 Structural analysis of Elongin C- pVHL interaction

Stebbins et al. first solved the crystal structure of pVHL in 1999. They showed that VHL has two domains: a roughly 100- residue NH2-terminal domain rich in β sheet

(β domain) and a smaller α-helical domain (α-domain), held together by two linkers and a polar interface (Fig.24).A large portion of the α-domain surface, and a small portion of the β domain, interacts with Elongin C. About half of the tumourigenic mutations map to the α domain and its residues that contact Elongin C [2]. The remaining mutations map to the β domain, and significantly, the author’s noted to a

βdomain surface patch uninvolved in Elongin C binding. This suggested that two macromolecular binding sites might be requiredfor the tumour suppressor effects of VHL. At this time HIF had not yet been identified as a VHL binding protein.

The β domain of VHL consists of a seven-stranded β sandwich (residues 63 to 154) and an α helix (H4; residues 193 to 204) that packs against one of the β sheets through hydrophobic interactions (Figs. 24 and 25). The α domain of VHL (residues 155 to 192) consists of three α helices (H1, H2, and H3). A helix from ElonginC (H4) completes the four-helix cluster arrangement, giving two pairs of helices packing at a perpendicular angle (Fig.24). The α and β domains are connected by two short

α domain β domain

Figure 24. The VCB ternary complex. On the left is a ribbon

diagram illustrating the secondary structure of the VCB complex. On the right is a topology diagram in which circles indicate helices and wide arrows indicate strands. C, COOH-terminus; N, NH2-terminus.

polypeptide linkers (residues 154 to 156and 189 to 194) and by a polar interface that is stabilised byhydrogen-bond networks from the H1 helix, the β sandwich, andElongin C. Several of the residues at the inter-domain interface have been found mutated in tumours.

The H1 helix of the VHL α domain coincides with the 12-amino acid segment shown to be important for Elongin C binding, and the structure reveals that it makes extensive contacts toElongin C. The most significant van der Waals contacts are made by Leu158, which protrudes from the H1 helix and fits into an Elongin C pocket, and by Cys162 and Arg161 (Fig.26). These are augmented by contacts from the Lys159, Val165, Val166, and Leu169 side chains of VHL.The other two helices of the α-domain also contribute contacts (Leu178_{, Ile}180_{, and Leu}184_{), with Leu}184

making the most extensive ones in this region. Additional contacts are made by residues in the first α-β linker (Leu153 _{and Val}155_{) and by Arg}82

from the β domain. The hydrogen bonds madeby Arg82, together with those made by Lys159 and Arg161 from the H1 helix, represent the few significant hydrogen-bond contacts made at the VHL-Elongin C interface. The arginine sidechains are also anchored in the hydrogen-bond networks of theVHL α-β domain interface.

Figure 26. The VHL-Elongin C interface consists of an intermolecular hydrophobic four-helix cluster

augmented by additional contacts. VHL and Elongin C

secondary structural elements are shown in red and blue, respectively. VHL amino acids are in yellow and those of Elongin C are in cyan. Hydrogen bonds are indicated by white dashed lines. Red atoms indicate oxygen and blue, nitrogen. A red circle with the letter "M" indicates a residue that is one of the six most frequently mutated in cancer. The Elongin C pocket where the VHL Leu158 binds is made up of Tyr76_{, Phe}93_{, Leu}103_{, and Ala}107_{, and}

Cys112.

Adapted from Stebbins et al. 1999

Figure 25. Sequence of VHL demonstrating that tumour-derived missense mutations are divided between the α and β domains of VHL, whereas residues contacting ElonginC cluster in the α domain. The histogram represents

279 mis-sense mutations in the database (Beraud et al. 1998). The six most frequently mutated amino acids are labelled. Shaded squares above each residue describe the relative solvent exposure of a residue in a hypothetical VHL

monomer. Blue boxes indicate residues that make hydrogen bonds or van der Waals contacts with Elongin C.

In addition the 40-amino acid SOCS (suppressor of cytokine signalling) -box sequence motif was shown to bind Elongin C and to contain sequence homology with the H1 helix of VHL. When the sequence of the SOCS1 SOCS box was compared to a library of 1,925 structures, the VHL α-domain ranked first [110, 111]. In this alignment, the pattern of hydrophobic residues in SOCS-1 and the SOCS-box consensus matches that of the entire VHL α-domain (Fig.27). These findings, in conjunction with the reported SOCS box-Elongin C binding data [110], indicate that the SOCS box and VHL α-domain represent a common structural and functional motif. Growing evidence suggests that the SOCS box, similar to the F-box of the SCF (Skp1-Cul1-F-box protein) complex, acts as a bridge between specific

substrate-binding domains and the more generic proteins comprise a large family of E3 ubiquitin protein ligases [112]. As discussed again in chapter 4, this sequence and structural homology helped unveil the first known function of pVHL as a component of an E3 ubiquitin ligase complex.

2.9.2 Structural analysis of HIF1α - pVHL interaction

The proposed model by Stebbins et al. that residues located within the β domain, a domain to which many mutations have been attributed, is an alternative macromolecular binding site required for tumour suppressor effects by VHL was confirmed structurally in a follow up paper by the Pavletich group in 2002 [113]. In this paper, Min et al. demonstrated that pVHL binds Hypoxia Inducible factor-1α (HIF-1α), and that this interaction is dependent on a modification involving molecular oxygen which results in the hydroxylation of a conserved Proline residue at position 564 within the HIF1α

sequence [113, 114]. The consequence of this interaction is targeting of HIF-1α for ubiquitin-mediated proteasomal degradation [114]. It is now also appreciated that a second proline hydroxylation event occurs at Pro-402 of HIF-1α. These sites contain a conserved LxxLAP motif and are targeted by a newly defined prolyl hydroxylase activity that in mammalian cells is provided by three isoforms termed PHD (prolyl hydroxylase domain) 1–3 (see chapter 4).

The structure illustrated in figure 28 shows that a 15-amino acid portion ofHIF-1α

(residues 561 to 575) adopts an extended, β strand-likeconformation. It binds pVHL in a Figure 27. Alignment of the VHL α-domain, the SOCS1 and SOCS-box

consensus, and the Skp2 and F-box consensus sequence. The VHL secondary

structure is shown above the alignment and the α-domain is indicated. Residues that stabilise the four-helix cluster are indicted in blue. Regions predicted to be α- helical are underlined in red.

site on pVHL. A six-residue N-terminal segment (residues 561 to 566) that is centred on Hyp564 _{(Hyp is the three-letter}

code for hydroxyproline), and a four-residue C-terminal segment (residues 571 to 574) are separatedby a four- amino acid bulge that does not contact pVHL (Fig.28.B). HIF-1α interacts exclusively with the β domain of pVHL. It

binds alongside the

βsandwich, making five backbone-backbone

hydrogen bonds. The side of the pVHL β sandwich where HIF-1α binds has the hydrophobic core partially

exposed. This exposed hydrophobic patch, together with several partially buried polar residues, makes up the binding site of the hydroxyproline. The hydroxyproline has a central role in complex formation. It is nearly entirely buried, with 96% of its accessible surfacearea in a hypothetical free peptide covered by pVHL. The pyrrolidinering inserts toward the partially exposed hydrophobic core of the pVHL β domain, making multiple van der Waals contacts with Trp88, Tyr98, and Trp117 of pVHL (Fig.29). The 4-hydroxyl group inserts farthest into pVHL and forms

hydrogen bonds with the Nδ of His115 and the OH group of Ser111, both of which also form hydrogen bonds with other pVHL groups. The pVHL residues that interact with Hyp564 are highly conserved in the human, mouse, frog, fly, and worm pVHL orthologues (Fig.28.B). Trp88, Tyr98, His115, and Trp117 are among the 11 β domain residues that are invariant in the five orthologues, and Ser111 is replaced with a threonine in the frog, fly, and worm. Mutations of Tyr98, Ser111, and Trp117 of pVHL have been shown to abolish HIF1α binding [115]. Compared with Hyp564, the other N-segment residues make significantly fewer contacts. Among them, Ile566 makes the most contacts,

Figure 29. The contacts made by the N segment, and in particular by Hyp564, are central to the binding of HIF1α to pVHL. The side chains of HIF1α and pVHL are coloured in light blue and yellow, respectively. The backbones of HIF1α and pVHL are in medium blue and red, respectively. The dotted lines indicate hydrogen bonds between the Gln67 Oδ1, Tyr98 Oη, His110 NH, and His110 CO groups of pVHL,

and the Leu562 _{NH, Hyp}564 _{CO, Tyr}565 _{NH, and Tyr}565 _CO

groups of HIF1α.

Adapted Min et al.2002

Figure 28. The HIF1α destruction sequence binds the β domain of pVHL. (A)

Schematic representation of the 15-residue portion of the HIF-1α destruction sequence bound to the β domain of pVHL in the pVHL-ElonginB-ElonginC complex. HIF1α is in blue, Hyp564

in yellow, pVHL in red, ElonginB in magenta, and ElonginC in green. (B) Alignment of the first destruction sequence (containing Hyp564) in the ODDs of HIF1α orthologues and HIF2α and HIF3α paralogues, highlighting residues identical in seven of the nine sequences. The putative N and C segments of the second destruction sequences

(containing Hyp402) of HIF1α and HIF2α orthologues are aligned below. The reported second destruction sequence is 38 residues long, and only a 23- residue region containing the conserved LxxLAP motif that aligns with the first destruction sequence is shown.

interacting with Pro99 and Ile109 of pVHL. Met561 packs with Phe91 of pVHL, Leu562 with Tyr112 and Arg69, Ala563 with Trp88, and Tyr565 with His110 (Fig.29). These side-chain contacts occur on the surfaceof the complex, however, and are unlikely to make a major contributionto specificity and affinity compared with Hyp564_.

A second,independent destruction sequence containing Hyp402 within the HIF-1α

ODD interacts with pVHL in a similar mode assuming seven insteadof four residues in the bulge between its putative hydroxyproline-containingN-segment- and C-segment-like sequences (Fig.28.B) [7]. This awaits further structural determination.