Nathaniel L. Hepowit1, Hugo V. Miranda1, Sivakumar Uthandi1,2, Alyssa Berganini1, Dieter Söll3,4, Markus Englert3, Nikita Nembhard1, and Julie A. Maupin-Furlow1,5

1Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700

Abstract

Introduction

Results

This work was funded in part by the NIH (ROI GM057498) and the DOE (DE-FG02-05ER1560) to Dr. Julie A. Maupin-Furlow. Special thanks to FORD IFP for the scholarship and travel grant (to UK for the Society for General Microbiology 2011 Sring Conference) accorded to the primary author.

Acknowledgment

Conclusions

Figure 7. UbaA in free (A) and SAMP-conjugated forms (B). Sampylation of UbaA on certain Lys residues (C) might regulate enzyme function and alter protein half-life. LC-MS/MS results show that SAMP1 modifies K157, whereas SAMP2 on K87 and K113 through an isopeptide bond (D). SAMP-mediated modification at K157 might block the SAMP-binding pocket of UbaA. Modified K87 might not bind ATP for SAMP adenylation, thus negatively regulating UbaA function.

Figure 6. UbaA mediates the conjugation of human Ub (A) and E. coli ThiS (B) in the H. volcanii host, indicating that UbaA has a broad selectivity to Ubl substrates. (C) The C-terminal GlyGly is required for ThiS conjugation to its target protein substrates.

Figure 2. (A ) Multiple amino acid sequence alignment of UbaA with eukaryotic E1 and bacterial E1-like protein ThiF. Conserved UbaA residues are predicted to be involved in SAMP adenylation ( ), zinc-binding ( ), and thiol-dependent linkage formation ( ). The presence of the P-loop motif suggests that UbaA cleaves ATP. (B) Crystal structure of SAMP1 and SAMP2 visualized by PyMol. Sequence alignment of the carboxyl end of Ub-fold proteins, showing the identical (blue) and similar (cyan) amino acids and the conserved GlyGly (red) at the terminus.

A B

B

A B

A B

AEROBIC ANAEROBIC

UbaA activates SAMP1 and SAMP2 by forming a thiol-dependent complex with these proteins. SAMPylation and SAMP-dependent sulfur transfer (to tRNA and MoCo) require the C-terminal GlyGly of SAMP and conserved residues at the active site of UbaA, such as ATP-binding K87 and D131, Zn2+-binding C171 and C245, and catalytic C188. UbaA N71 is crucial in sulfur transfer but not in protein conjugation (SAMPylation).

UbaA is the archaeal counterpart of eukaryal E1 and bacterial MoeB/ThiF.

UbaA is required for sampylation, anaerobic growth (with DMSO as terminal electron acceptor), and tRNALysUUU thiolation, thereby providing a direct link between protein conjugation and sulfur transfer.

SAMP1 and SAMP2 can modify UbaA which could be a mechanism in regulating UbaA function and turnover.

3Department of Molecular Biophysics and Biochemistry, and 4Department of Chemistry, Yale University, New Haven, CT 06511

K157

K113K211

K87

SAMP GlyGly ~ UbaACys188

Cys171

Lys87 ATP

Zn2+

C

10-

15-

20-

25-

37-

50-75-

100-150-

kDa- empty

vector

Ub - UbaA

- Ub free

- Ubconjugates

WT C188S

ΔubaA genome

in trans

IB: α-FLAG

25-

37-

50-

- UbaA-StrepII

IB: α-StrepII

10-

15-

20-

25-

37-

50-75-

100-150-

kDa

ΔsamP1ΔsamP2Δsamp3

- emptyvector Ub Ub - empty

vector Ub Ub

ΔubaA ΔsamP1ΔsamP2Δsamp3 ΔubaA

- DMSO + DMSO

- Ub free

- Ub-conjugates

10-

15-

20-

25-

37-

50-75-

100-150-

kDa

ΔsamP1ΔsamP2Δsamp3

- emptyvector ThiS ThiS - empty

vector ThiS ThiS

ΔubaA ΔsamP1ΔsamP2Δsamp3 ΔubaA

- DMSO + DMSO

- ThiS free

- ThiS-conjugates

150-

kDa - emptyvector

ThiS - UbaA

WT C188S

genome

in trans

10-

15-

20-

25-

37-

50-

75-

100-

- ThiS-conjugates

- ThiS free

ΔubaA

25-

37-

50-

- UbaA-StrepII

IB: α-FLAG

IB: α-StrepII

10-

15-

20-25-

37-50-75-

150-

kD

genome

in transemptyvector ThiS GG ThiS

- ThiS free

- ThiS conjugates

samp1 samp2 samp3C

A B adenylation Zn2+ coordinationThioester linkage

other Cys adenylation Zn2+ coordinationThioester linkage

other Cys

Ubiquitin

PDB: 1FXT

GlyGly

PDB: 1ZUD

GlyGly

ThiS

Modified from Dahl et al. (2011). JBC.

Figure 8. (A) Modeled structure of UbaA-SAMP1 complex showing conserved residues involved in SAMP adenylation (N71, K87, D131 and R136) and zinc-binding (C171, C174, C245 and C248). C188 is believed to form a thiol-dependent linkage with the Gly-carboxyl terminus of SAMP immediately after the release of AMP from SAMP adenylate. This predicted structure is modeled after the crystal structure of MoeB-MoaD (PDB 1JW9) of Escherichia coli. (B) Proposed model of the archaeal Ubl system showing that the activating enzyme UbaA is required in both SAMP conjugation and sulfur coordination.

Figure 1. Ubiquitin-fold proteins, which highly vary in amino acid sequence, characteristically adapt the β-grasp fold tertiary structure and have the conserved GlyGly at their carboxyl termini. Ub-fold proteins Ub, MoaD, and ThiS are involved in protein conjugation, molybdopterin biosynthesis, and tRNA thiolation, respectively.

N71

ATP

D131

K87

R136

C188

C174

C171

C245

C248

UbaA

SAMP1

Zn2+

PDB: 3PO0

SAMP1

GlyGly

SAMP2

GlyGlyPredicted Structure

MoaD

GlyGly

PDB: 1JWA

FORD IFP

A

2Tamil Nadu Agricultural University, Coimbatore 641 003, India

5Genetics Institute, University of Florida, Gainesville, FL 32611-0700

Ubiquitylation, a posttranslational conjugation of ubiquitin (Ub) to target protein substrates, is a universal key regulatory mechanism in various cellular processes in eukaryotes. In bacteria, the ubiquitin-like (Ubl) proteins MoaD and ThiS are not involved in protein conjugation but rather serve as sulfur donor in molybdopterin-based cofactor (MoCo) biosynthesis and tRNA thiolation, respectively. Prior to conjugation or sulfur transfer, Ub-fold proteins Ub, MoaD, and ThiS require adenylation by activating enzymes E1, MoeB, and ThiF, respectively. Recently, we found that the archaeal Ubl proteins (SAMP1 and SAMP2) are both protein modifiers and sulfur donors; however, the mechanism of SAMP activation has not been elucidated. Here we identify the archaeal activating enzyme UbaA, a protein structurally homologous and functionally analogous to E1/MoeB/ThiF, required for sampylation and SAMP-mediated thiolation, thereby linking the two divergent biochemical pathways - protein conjugation and sulfur transfer. Two alternative procedures were employed in characterizing UbaA function: (i) separate recombinant expression and purification of His6-UbaA and Flag-His6-SAMP and subsequent reconstitution of activation products in vitro, and (ii) co-expression of in trans Flag-SAMP and UbaA-StrepII in wildtype and ΔubaA knockout hosts for analysis of notable phenotypes. In the first approach, we detected by site-directed mutagenesis and immunobloting that the formation of thiol-dependent UbaA~SAMP complex requires ATP and a metal cofactor. The conserved K87 was found crucial for ATP binding and UbaA~SAMP complex formation. Coupled with deletion experiments, it is suggested that a thioester bond bridges the C-terminal GlyGly of SAMP and the conserved catalytic Cys (C188) of UbaA. Site-directed mutation of UbaA residues K87 and D131 (predicted to bind ATP), C171 and C245 (predicted to bind Zn2+) and C188 (the conserved catalytic cysteine) was found to inhibit SAMPylation and sulfur transfer (based on indirect assay for tRNA thiolation and MoCo biosynthesis). In contrast, UbaA N71, modeled and predicted to be involved in SAMP adenylation, was found to be crucial for sulfur transfer but not for sampylation. Interestingly, UbaA has a broad selectivity to Ub-fold substrates, including human Ub and Escherichia coli ThiS, which further corroborate the functional evolutionary relationship between eukaryotic protein modifiers and bacterial sulfur donors. Furthermore, immunoblot and LC-MS/MS analyses show that K87, K113, and K157 of UbaA can be modified through formation of an isopeptide bond with SAMP1 or SAMP2. Thus, it is proposed that sampylation might regulate UbaA function and alter its half-life by proteasome-mediated turnover. Overall, this study provides a fundamental insight into the diverse cellular functions of the Ubl system especially in understanding the central role of Ubl activation, which determines specific downstream conjugation pathways and sulfur coordination.

Figure 4. In vivo SAMPylation was carried out by co-expressing Flag-SAMP1 (A) or Flag-SAMP2 (B) with UbaA (wildtype or site-directed mutants) in H. volcanii ΔubaA grown in glycerol minimal medium supplemented with Ala. Whole-cell lysates normalized to 0.065 OD600 were resolved on SDS-PAGE gel and visualized by Western Blotting. Similar with E1 and other E1-like proteins, results suggest that ATP-binding K87, D131, zinc-binding C171 and C245, and conserved catalytic C188 of UbaA are indispensable for SAMP1 and SAMP2 conjugation in nitrogen-limited medium.

Figure 5. UbaA is not only essential in anaerobic growth of H. volcanii (in the presence of DMSO as terminal electron acceptor)(A) but also in tRNALysUUU thiolation (B). MoCo-requiring DMSO reductase converts DMSO to dimethyl sulfide in the absence of O2. As we proposed previously, UbaA-activated SAMP1 and SAMP2 incorporate sulfur to MoCo and tRNA, respectively. Together with Fig.2, N71 is essential only in sulfur transfer but not in SAMPylation.

Figure 3. UbaA activates SAMP1 (A) and SAMP2 (B) by forming a ~50 kDa thiol-dependent UbaA~SAMP complex in the presence of ATP. In vitro activation of SAMP by UbaA was performed in a reaction containing 2.5 µM UbaA, 5.0 µM SAMP2, 5.0 mM ATP, 5.0 mM MgCl2, 2 M NaCl, 1 mM DTT, and 20 mM HEPES, pH 7.5. (C) UbaA~SAMP complex formation requires K87 (ATP-binding), C188 (catalytic Cys), and C171 (Zn+2 coordination).

AEROBIC ANAEROBIC

IB: α-FLAG IB: α-FLAG IB: α-FLAG

ΔubaA: Flag-SAMP1—UbaA-StrepII

ΔubaA: Flag-SAMP2—UbaA-StrepII

IB: α-StrepII IB: α-StrepII IB: α-FLAG IB: α-FLAG

- UbaA

Δsamp1Δsamp2Δsamp3: UbaA-StrepII

IB: α-StrepII

37-

50-

25-

kDa

UbaA

A B Proposed Model of the Archaeal Ubl System

C VDK(gg)SNVHEVVAGSDVVV

K113

M/Z-933.9823,2+

D

EUK

AR

YA

BA

CTE

RIA

A B

Crystal Structure

P-loop

Archaeal activating enzyme (UbaA) has broad selectivity to substrates, including eukaryal and bacterial Ub-fold proteins.