Generation of E2-binding site mutants 1 Potential amino acids to be mutated

In the previous chapter, a series of key residues in UBE2D2 were identified as being potentially important for interaction with OTUB1. These residues were identified by overlaying spectrum of the 15N-UBE2D2:ΔNOTUB1 complex and an available qualitative UBE2D2 NMR spectrum (Houben et al., 2004). However, predicted binding points may not be exact due to the parameter differences used to record both spectra. Also, observed shifts may be the consequence of binding rather than actual physical points of contact.

Targeted mutagenesis was performed to generate forms of UBE2D2 with amino acid point mutations in order to determine if amino acid substitution is sufficient to disrupt interaction with OTUB1, ΔNOTUB1 or E3-RING proteins. If this is the case then it would implicate these particular amino acids as being necessary for binding to OTUB1, thereby validating our UBE2D2 binding interface predictions. Therefore, Lys8, Glu9 and Asp12 within the 1st α-helix and Lys101 at the L2 loop of 4th

β-strand and H2 α-helix were chosen to be mutated as they are all polar, and polar amino acid side chains tend to gather on the outside of the protein where they have the potential to interact with the aqueous environment. Meanwhile, the non-polar amino acid side chains are buried on the inside to form a tightly packed hydrophobic core of atoms that are hidden from the aqueous environment (Alberts et al., 2002). For this reason, we avoided mutating non-polar amino acids in order to avoid imposing unwanted conformational changes. Also, polar amino acids are more likely to be accessible on partner binding surfaces.

Acidic side chain amino acids Glu9 and Asp12 were mutated to alanine or arginine respectively. As the simplest and smallest amino acid, mutation to alanine could ensure that the mutations will not disrupt the secondary structure due to stearic hindrance (Alberts et al., 2002). It was also thought to be a good idea to mutate them into basic side chain amino acids such as lysine, arginine or histidine in order to swap the acidity/basicity in order to see if the

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binding interaction could be abolished rather than substituting them into another acidic amino acids, which may also mediate interactions. Therefore, Glu9 was substituted to Ala (E9AUBE2D2) and Asp12 was changed to Arg (D12RUBE2D2). Meanwhile, both Lys8 and Lys101 were mutated to alanine (K8AUBE2D2 and K101AUBE2D2). All these residues were mutated following QuikChange® site-directed mutagenesis protocol.

5.2.2 Mutagenesis strategy

QuikChange® mutagenesis was carried out as described in Chapter 2 and point mutations were made in such a way that encoded amino acids were converted to either alanine or arginine. Two complementary forward and reverse primers were designed carrying base substitutions in the centre of the primers as tabulated below:

Table 5.1 Forward and reverse UBE2D2 mutagenesis primers: The mutation conferred by these primers is described in the “Mutation” column. Bases that are mutated compared to the wild-type sequence are highlighted in maroon and pink.

Mutation Forward primer Reverse primer

1 K8A 5’ G AAG AGA ATC CAC GCC

GAA TTG AAT GAT CTG 3’

5’ CAG ATC ATT CAA TTC GGC

GTG GAT TCT CTT C 3’

2 E9A 5’ GA ATC CAC AAG GCC TTG

AAT GAT CTG G 3’

5’ C CAG ATC ATT CAA GGC

CTT GTG GAT TC 3’ 3 D12R 5’ G GAA TTG AAT CGC CTG GCA CGG G 3’ 5’ C CCG TGC CAG GCG ATT CAA TTC C 3’ 4 K101A

5’ GCA CTA ACT ATT TCA

GCC GTA CTC TTG TCC

ATC 3’

5’ GAT GGA CAA GAG TAC GGC

TGA AAT AGT TAG TGC 3’

5 K8A,E9A 5’ GA ATC CAC GCCGCC TTG

AAT GAT CTG G 3’

5’ C CAG ATC ATT CAA GGC

GGC GTG GAT TC 3’

6 K8A,D12R 5’ G AAG AGA ATC CAC GCC

GAA TTG AAT CGC CTG 3’

5’ CAG GCG ATT CAA TTC GGC

GTG GAT TCT CTT C 3’

7 E9A,D12R 5’ GA ATC CAC AAG GCC TTG

AAT CGC CTG G 3’

5’ C CAG GCG ATT CAA GGC

GGC GTG GAT TC 3’ 8 K8A,E9A, D12R 5’ C GCC TTG AAT CGC CTG GCA CGG G 3’ 5’ C CCG TGC CAG GCG ATT CAA GGC G 3’

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The mutation strategy was to perform PCR-amplification firstly using primers 1, 2, 3 and 4 on UBE2D2 in pDONR207 in order to generate a series of point UBE2D2 mutants: K8A

UBE2D2, E9AUBE2D2, D12RUBE2D2 and K101AUBE2D2. PCR for double point mutations could then be performed in these single mutants using the appropriate primers. The PCR products were treated with DpnI before transforming them into chemically competent cells for positive transformants selection. DpnI is a restriction endonuclease that recognises a short DNA sequence (GATC), but only when the DNA is methylated. DNA replicated within bacteria, like the template used in the reactions, will be methylated and therefore recognised by DpnI which will subsequently cleaved it. However, products synthesised in vitro will not be methylated and will not serve a substrate. They will be safely transformed into bacterial cells as an intact vector containing the UBE2D2 mutants ORF.

This experiment aimed to generate UBE2D2 as single, double, triple and quadruple mutants, however only 8 mutants were successfully generated. These included all the single mutants K8A

UBE2D2, E9AUBE2D2, D12RUBE2D2 and K101AUBE2D2, and the double mutants K8A,K101A

UBE2D2, K8A,E9AUBE2D2, K8A,D12RUBE2D2 and E9A,D12RUBE2D2. Generation of the mutants proved to be quite problematic and involved many failed attempts to produce positive transformants. This might be due to various factors for instance, an older mutagenesis kit was initially being used so there is a possibility that the enzymes may have denatured or not had their full activity. In addition, other solutions such as the reaction buffer or dNTPs may also not be as active due to numerous freeze-thaw cycles. Mutagenesis was therefore repeated using a new kit. The transformation was originally performed into XL 10- Gold Ultracompetent cells and then changed to XL 1-Blue Supercompetent cells in an attempt to increase the transformation efficiency. Optimisations were also made to the volume of DpnI-treated DNA transformed into cells and also the transformation solution that was plated to increase the probability of bacterial colony growth. The generation of other double, triple and quadruple mutants was undertaken, however due to time constraints were not produced. Nevertheless, it would be interesting if all the remaining double mutants and

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the triple and quadruple mutants could be produced in order to analyse further the importance of the binding sites.

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