Initial NMR analysis was performed to identify the binding interface of E2s and wild type (WT) OTUB1 complexes. Specific chemical shift patterns were observed in these studies suggesting specific points of contact between these two proteins (discussed further in Chapter 4). However, the full range of amino acids involved in these interactions could not be determined due to a very dense central region of the spectrum caused by an intrinsically disordered region within the OTUB1 protein. These regions (termed ‘spaghetti’ because they usually exist as unorganised, flexible short linear peptide motifs) do not form a well-defined three-dimensional structure and sometimes even appear to be totally unfolded in their native state. As these types of regions can affect protein solubility and crystallisability which were an important part of our proposed plans, the probability disorder for OTUB1 was calculated using RONN (Yang et al., 2005), which decides the likelihood of disorder based on alignments to a group of sequences of known folding state (Figure 3.11).
Figure 3.11 Probability of disorder for OTUB1: The amino acids 1-18, 48-49, 59- 71, 116-126, 235-248 are above probability cut-off of 0.5 and considered to be unfolded.
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RONN predicted a strong area of disorder at the N-terminus of OTUB1, which does not include the catalytic site. In addition, in structural studies performed on the OTUB1 protein, the N-terminal region was removed to produce OTUB1 (residue 40-271 only) crystal which successfully diffracted to 1.7Å resolution (Edelmann et al., 2009). Therefore, assuming that the N-terminal region would be problematic for structural studies we generated the truncated form of OTUB1 which lacked the N-terminal (1-39) amino acids.
It was possible that removal of the N-terminal region of OTUB1 could make OTUB1 more like OTUB2, as the main difference between the two proteins is the presence of the N- terminal extension in OTUB1 (Figure 3.12). Significantly, no interaction was observed between OTUB2 and any E2 proteins in our screens. In order establish if N-terminally truncated OTUB1 could still interact with defined E2 partners, further Y2H screens were performed using newly generated ΔNOTUB1 bait and prey constructs.
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Figure 3.12 Superposition of OTUB1 and OTUB2: A very similar display with identical folds of OTUB1 (40-271) without N-terminal (PDB ID: 2ZFY) and OTUB2 (PDB ID: 1TFF). Picture generated using MacPyMOL v1.3.
OTUB1
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3.5.1 Generating ΔNOTUB1
To generate ΔNOTUB1 Y2H bait and prey constructs, PCR was performed on full-length OTUB1 in pDONR223 using a gene-specific forward primer instead of vector-specific primer as used previously. The gene-specific primer was designed to contain a sequence of OTUB1 starting from base 118 (blue font) following the yeast/Gateway® sequence (red font):
5’ GAATTCACAAGTTTGTACAAAAAAGCAGGCTGGATGGAGATTGCTGTGC 3’.
This primer would anneal in such a way as to amplify from base 118 onwards hence 117 bases representing the first 39 amino acids were removed. The same reverse primer pDONR223 GR R1 was used in conjunction with the newly designed forward primer. The product of this PCR is N-terminal truncated OTUB1 (aa 40-271) with overhang gap repair sequence at the 5’ and 3’. Consequently, gap repair transformation was performed to using this PCR product to generate bait clones in pGBAE-B/Mat-a cells and equivalent prey clones in pACTBE-B/Mat-α. Following YC-PCR analysis of gap repair colonies, verified clones were tested for autoactivation.
3.5.2 ΔNOTUB1 slightly reduced interaction with E2 binding partners
Y2H results show that removal of N-terminal did not totally abolished interactions with E2 proteins (Figure 3.13). However, the interaction profile between ΔNOTUB1 and its binding partners were greatly reduced especially in the ΔNOTUB1 prey orientation, which might be due to a stearic effect. The stearic effects arise from the fact that each atom within a molecule occupies a certain amount of space. In some cases, if atoms are brought too close together, this may affect the molecule's preferred conformation and reactivity. A truncation is commonly disfavoured and notoriously known to cause stearic interaction with the fusion domain resulting in a reduced ability to bind to known interaction partners. The ΔNOTUB1 Y2H screen were analysed and the candidates for structural analysis were selected upon evaluating Y2H data.
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(A)
OTUB1 FL (+ve control) ΔNOTUB1
-W LH (3 -A T) -W LA (B) 1 2 3 4 5 6 7 8 9 10 11 12 A UBE2A UBE2B UBE2C UBE
2D1 UBE2D2 UBE2D3 UBE 2D4 UBE 2E1 UBE 2E3 UBE 2G1 UBE 2G2 UBE2H B UBE2I UBE2L3 UBE2L3 UBE2L6 UBE2M UBE2N UBE2V1 UBE2V2 UBE2S UBE2R1 UBE2K UBE2R2 C UBE2J1 UBE2J2 UBE2W UBE
2E2 UEVLD UEVLD UBE2W UBE2Z AKTIP UBE2O UBE2Q1 UBE2T D UBE2I UBE2F BIRC6 UBE2U UBE2DNL UBE2Q2 UBE2Z TSG101
(C)
OTUB1 FL (+ve control) ΔNOTUB1
-W LH (3 -A T) -W LA (D) 1 2 3 4 5 6 7 8 9 10 11 12 A UBE2A UBE2B UBE2C UBE
2D1 UBE2D2 UBE2D3 UBE2D4 UBE2E1 UBE2E3 UBE
2G1 UBE2G2 UBE2H B UBE2I UBE2L3 UBE2L3 UBE2L6 UBE2M UBE2N UBE2V1 UBE2V2 UBE2S UBE2R1 UBE2K UBE2R2 C UBE2J1 UBE2W UBE2E2 UBE2W UBE2Z AKTIP UBE2O UBE2Q1 UBE2T UBE2I UBE2F BIRC6 D UBE2U UBE2W
Figure 3.13 ΔNOTUB1:E2 interactions: (A) ΔNOTUB1 prey mated against E2 baits on SD-WLA and SD-WLH(3-AT) at day seven. (B) Grid positions of the E2 baits assayed in Figure 3.13(A). (C) ΔNOTUB1 bait mated against E2 preys on SD-WLA and SD-WLH(3- AT) at day 7. (D) Grid positions of the E2 preys assayed in Figure 3.13(C).
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