Multiple protein sequence alignment

65

FAT10 (Aichem et al., 2010) was co-transformed with pGADT7-NUB1L in the NMY51 yeast strain.

2.6.2.2 Multiple protein sequence alignment

The amino acid sequence of the VWA domain of hRpn10 of 26S proteasome from different organisms was obtained from Conserved Domains Database (Marchler-Bauer et al., 2011)and were aligned and formatted using Jalview (Waterhouse et al., 2009) and ALINE (Bond and Schuttelkopf, 2009). The percent identity was calculated using Jalview. The alignments for N- and C-terminal FAT10 with ubiquitin were performed using CLUSTAL W (Thompson et al., 1994) and formatted using ALINE.

Proteasomal targeting of FAT10 by the VWA domain of hRpn10 and NUB1L  2.

Supplementary Table 2.1 Yeast strains. The table representing the yeast strains used in this study along with the designation and genotype.

Strains Genotype References

AH109 MATa, trp1-901, leu2-3,112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA -HIS3,GAL2UAS-GAL2TATA-ADE2,

URA3::MEL1UAS-MEL1TATA-lacZ, MEL1

Clontech

NMY51 MATa his3Δ200 trp1-901 leu2-3,112 LYS2::(lexAop)4-HIS3 ura3::(lexAop)8-lacZ (lexAop)8-ADE2 GAL4

Dualsystems Biotech

MHY500 MATa, his3-Δ200, leu2-3,112 ura3-52, lys2-801 trp1-1

(Chen et al., 1993)

NRY5 MHY500 with rpn10Δ::kanMX6 This study NRY51 MHY500 with pdr5Δ::hphMX4 This study NRY53 MHY500 with rpn10Δ::kanMX6, pdr5Δ::hphMX4 This study

WCG4a MATa, his3-11,15 leu2-3,112 ura3 Can^S GAL2 (Heinemeyer et al., 1997)

YHI29/1 pre1-1 (Palanimurugan et

al., 2004)

Proteasomal targeting of FAT10 by the VWA domain of hRpn10 and NUB1L  2.

67

Supplementary Table 2.2 Gene cloning. The table showing the genes cloned into different vectors along with the restriction sites used.

Insert Vector Restriction site Reference for vectors FAT10 pGADT7 BamHI and XhoI Clontech Laboratories NUB1L pGADT7 NdeI and ClaI Clontech Laboratories hRpn10 YEplac181 EcoRI and KpnI (Gietz and Sugino, 1988)

VWA pET26b NdeI and HindIII Novagen

VWA YEplac181 EcoRI and KpnI ---

UIM1UIM2 YEplac181 EcoRI and KpnI --- hRpn10 (UIMs

mutated)

YEplac181 EcoRI and KpnI ---

FAT10 p416Met25 SpeI and SalI (Mumberg et al., 1994) NUB1L p414GPD SpeI and SalI (Mumberg et al., 1994) scRpn10 pET45b(+) KpnI and HindIII Novagen

NUB1LΔUBA pGEX2TKS NotI and XbaI GE Healthcare (pGEX2TK modified) NUB1LΔUBL pGEX2TKS NotI and XbaI ---

FAT10 K0 p416Met25 SpeI and SalI --- Rpn10ΔD11 YEplac181 EcoRI and KpnI --- hRpn10ΔD11 YEplac181 EcoRI and KpnI ---

3. Studies on the Structure of the hRpn10 Subunit of the 26S Proteasome and the

UBL-UBA Domain Protein NUB1L

Neha Rani, Tancred Frickey, Antje Schafer, Hermann Schindelin and

Marcus Groettrup

Studies on the Structure of hRpn10 and NUB1L  3.

69

3.1 INTRODUCTION

The major ubiquitin receptor in the 26S proteasome was recognized as Rpn10 which initiates the degradation of proteins conjugated to polyubiquitin chains (Deveraux et al., 1995a; Ferrell et al., 1996; Voges et al., 1999). The Rpn10 subunit also interacts with proteins containing UBL/UBA domains like Rad23/hHR23a (yeast/human) and Dsk2/PLIC1 (yeast/human) via the UBL domain (Funakoshi et al., 2002; Hiyama et al., 1999; Schauber et al., 1998; Walters et al., 2002), whereas another UBA domain containing protein p62 (sequestosome-1) interacts via the PB1 domain (which is similar to the UBL domain as it forms a ubiquitin-like β-grasp fold) (Bjorkqvist et al., 2008). The hRpn10 subunit is characterized by three domains, namely, the N-terminal von Willebrand A (VWA) domain and two C-terminal ubiquitin-interacting motifs (UIM1 and UIM2) (Hofmann and Falquet, 2001; Whittaker and Hynes, 2002; Zhang et al., 2009).

The UIM domain is composed of ~20 amino acids with α-helical structure. It is usually found in proteins involved in ubiquitylation and ubiquitin metabolism, or known to interact with ubiquitin-like modifiers (Hofmann and Falquet, 2001). The VWA domain is characterized by Rossmann folds consisting of β-sheet sandwiched by multiple α-helices.

The domain is found in extracellular matrix proteins and in integrin receptors, and usually involved in cell adhesion (Whittaker and Hynes, 2002). One of the most important noncontiguous sequence motif in this domain is a metal-ion dependent adhesion site (MIDAS) (D-x-S-x-S….T.…D) which is involved in its binding to metal ions (Whittaker and Hynes, 2002). The amino acid sequence alignment of this domain in hRpn10 subunit in various orthologs is shown in the Supplementary Figure 2.2 (Chapter 2).

Polyubiquitin interacts with hRpn10 through UIMs (Beal et al., 1998) but in our previous studies we observed that the VWA domain of hRpn10 interacts with FAT10 (Figure 2.2e, Chapter 2). NUB1L, a non-covalent interaction partner of FAT10, also binds to the VWA domain of the hRpn10 subunit (Figure 2.2e, Chapter 2). NUB1L is composed of a N-terminal UBL domain and three C-N-terminal UBA domains (Tanaka et al., 2003). UBL domains mimic ubiquitin in function and structure. They also form a β-grasp fold with an α-helix similar to ubiquitin. They play an essential role in the interaction with the 26S proteasome (Hartmann-Petersen and Gordon, 2004; Hochstrasser, 2009; Jentsch and Pyrowolakis, 2000; Su and Lau, 2009). The UBA domain consists of approximately 45 amino acids and is found in diverse proteins involved in the ubiquitin-proteasome

Studies on the Structure of hRpn10 and NUB1L  3.

pathway. It is characterized by relatively poor sequence conservation (Hofmann and Bucher, 1996). It comprises of a compact bundle of three α-helices and a small conserved hydrophobic surface formed by the C-terminus of α-helix 1, loop 1, and α-helix 3 (Madura, 2002). There are functional differences between the UBA domain proteins (Withers-Ward et al., 2000) suggesting that the hydrophobic surface may be a more general protein–protein interaction module. Amino acid residues, which are conserved, contribute to the structural similarity of UBA domains whereas diverse residues allow the interaction with different proteins. The alignment of representative UBA domains in the protein database as obtained by SMART analysis shows several consensus sequences (Figure 3.1 and Figure 3.2).

Various bioinformatics prediction, analysis and modeling tools can be utilized to generate hypotheses and theoretical models. The most extensively employed application is to derive functional information about proteins. Another important application includes designing site-directed mutagenesis experiments and thereby predicting ligand binding site(s). The protein data bank (PDB) is widely used as a source of information about the structure of proteins, both experimentally and theoretically. The protein structure prediction methods are basically classified into two categories: (a) comparative modeling methods predict the structure based on the known protein structures (Bowie et al., 1991;

Sali and Blundell, 1993), which could be either a sequence-sequence comparison based approach (Karplus et al., 1998) or a sequence-structure comparison based approach (threading) (Bowie et al., 1991; Jones et al., 1992); (b) ab initio methods predict structures directly from the protein sequence (Li and Scheraga, 1987; Skolnick and Kolinski, 1991). One of the most reliable software for predicting the three-dimensional structure of proteins is HHpred (Soding et al., 2005), which is based on the identification of homologous sequences with the known structures by searching the local or global alignment using alignment database like Pfam (Sonnhammer et al., 1998) or SMART (Ponting et al., 1999; Schultz et al., 1998). This method works quite well because the structural changes are slow during evolution as compared to sequences and therefore, homologous proteins may have a similar structure even if their sequences have diverged.

This is the first server which utilizes the pairwise comparison of hidden Markov models (HMMs), which improves the sensitivity. The program calculates the three-dimensional structure using MODELLER software (Sali and Blundell, 1993).

Studies on the Structure of hRpn10 and NUB1L  3.

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Figure 3.1 Multiple sequence alignment of UBA domains representing the consensus amino acid residues. Multiple sequence alignment of UBA domains in 45 homologous proteins, as available on the SMART server. The high degree consensus sequences are highlighted. Proteins are represented on the left side by their PDB IDs.

Studies on the Structure of hRpn10 and NUB1L  3.

Figure 3.2 Hidden Markov Model Logo of UBA domains representing the consensus amino acid residues. The logo was obtained using SMART software based on an alignment of 45 homologous protein sequences bearing UBA domains. The total width of the red-shaped (dark and light) stack visualizes the expected number of inserted letters in alignment. The total stack height is computed as the information content of the column. The relative height of each letter within the stack is proportional to its frequency at that position. The highly conserved residues shown are MGF…AL..A…L.

In this study we employed comparative modeling methods to predict structures of the VWA domain of the hRpn10 subunit and NUB1L. The prediction of amino acid residues, involved in the interaction with FAT10 and NUB1L, was based on the modeled structure and the conservation pattern of VWA domains. These insights were validated by performing site-directed mutagenesis experiments. Unfortunately, all mutations analyzed theoretically as well as experimentally were not important for the interaction. Therefore, another approach was employed to answer this question, which involved obtaining the structure of the VWA domain by X-ray crystallography. To achieve this, we expressed the VWA protein in E.coli strain using SUMO fusion technology (Panavas et al., 2009).

The purification was accomplished by using affinity and gel filtration chromatography.

Similarly, we also purified full-length hRpn10 protein by utilizing affinity and ion-exchange chromatography. Currently, we are scaling up the purification of protein to obtain high yield of protein. This study will give an insight into the new interaction site on the VWA domain of the hRpn10 subunit which has never been reported.

On the other hand, we also modeled the structure of NUB1L and strikingly, observed that NUB1L is composed of four UBA domains instead of three. The alignment of the newly identified domain with the previously known UBA domains confirmed our structural

Studies on the Structure of hRpn10 and NUB1L