Aminoacyl tRNA Synthetases in Translation

GTPGTP

Elongation factor

(EF)

Aminoacyl-tRNA

(AA-tRNA)

Aminoacyl-tRNA

(AA-tRNA)

Ribosome

Growing Protein Chain

Messenger RNA

(mRNA)

Ribosome

Growing Protein Chain

Messenger RNA

(mRNA)

Ribosome

Growing Protein Chain

Messenger RNA

(mRNA)

Aminoacyl-tRNA Synthetase (ARS)

+

Amino Acid

Transfer RNA (tRNA)

ATP

5'3'

Aminoacyl-tRNA Synthetase (ARS)

+

Amino Acid

Transfer RNA (tRNA)

ATP

5'3'

Aminoacylation of tRNA is a Two-step Reaction

N

NN

N

NH2

O

OHOH

OPO

O-

O

PO

O-

O

P-O

O-

O

+ +

ProRS Proline ATP

-PPi

ProRS•Pro-AMP(Aminoacyl-adenylate)

+H2N

C O-

ON

NN

N

NH2

O

OHOH

OP

O-

O

+H2NC O

ON

NN

N

NH2

O

OHOH

OP

O-

O

+H2NC O

O

Step 1. Activation of the amino acid. ARS activates an amino acid in the presence of ATP to form the aminoacyl-adenylate intermediate:

Step 2. Amino acid is transferred to 3′-end of tRNA

+

O

OHOH

OA76

tRNAPro

O

OHOH

OA76

tRNAPro

-AMP

N

NN

N

NH2

O

OHOH

OP

O-

O

+H2NC O

OO

OHO

OA76

+H2N

CO

O

OHO

OA76

+H2N

CO

Pro-tRNAPro

3′5′

3′5′

Long-range Communications in Bacterial Prolyl-tRNA Synthetases

Proofreading reaction to remove non-cognate amino acid attached to tRNA

Editing domain

Catalytic domain

tRNA binding domain

Binds amino acid and ATP to form an activated intermediate known as amino acid adenylate

Binds specific tRNA and orients it towards the catalytic domain

A cartoon diagram of the structure of Enterococcus faecalies prolyl-tRNA synthetase (ProRS) (3). ProRSs from all three kingdoms of life misactivate non-cognate alanine and form alanyl-tRNAPro. Editing domain of bacterial ProRSs selectively hydrolyzes alanyl-tRNAPro (4).(3 Crepin, T., Yaremchuk, A., Tukalo, M., and Cusack, S. (2006), Structure 14, 1511-1525; 4 Wong, F. C., Beuning, P. J., Nagan, M., Shiba, K., and Musier-Forsyth, K. (2002), Biochemistry 41, 7108-7115.)

AbstractAminoacyl tRNA synthetases (ARSs) are an important family of protein enzymes that play a key role in protein biosynthesis. ARSs catalyze the covalent attachment of amino acids to their cognate transfer RNA (tRNA). They are multi-domain proteins, with domains that have distinct roles in aminoacylation of tRNA. Various domains of an aminoacyl-tRNA synthetase perform their specific task in a highly coordinated manner. The coordination of their function, therefore, requires communication between the domains. Evidence of domain-domain communications in ARSs has been obtained by various biochemical and structural studies (1). However, the molecular mechanism of signal propagation from one domain to another domain in ARSs has remained poorly understood. In the present work, we investigated the molecular basis of long-range domain-domain communication in Escherichia coli prolyl-tRNA synthetase (E. coli ProRS). In particular, we explored if an evolutionarily conserved and energetically coupled network of residues are involved in domain-domain signal transmission in E. coli ProRS. In this work, a combination of bioinformatics and biochemical methods have been employed to identify networks of residues involved in the long-range communication pathway. Initial results demonstrate that sparse networks of evolutionarily conserved and energetically coupled residues, located at the domain-domain interface, might have a significant role in long-range interdomaincommunications in Ec ProRS.

(1 Alexander, R. W., and Schimmel, P. (2001), Prog. Nucleic Acid Res. Mol. Biol. 69, 317-349.)

Evolutionarily Conserved or Coupled Residues Constitute a Sparse but Contiguous Network of Interactions

The evolutionarily conserved or coupled residues of E. coli ProRS are involved in the interaction networks. a) The conserved residues are indicated as red balls and labeled; the statistically coupled residue network has been shown as an ice-blue patch; b) A part of the inter-domain region (between the editing and the catalytic) is dominated by ionic interactions; hydrogen atoms are omitted for clarity. Alanine scanning mutagenesis has been performed to analyze the effect of mutation on enzyme function. Eight mutants (F147A, G217A, E218A, Y229A, R299A, H302A, K308A, and F359A) of E. coli ProRS were obtained by site-directed mutagenesis.

G217E218

R299

K308Y229

a) b)

R299

K308

H302

R388

D394

E303

K279

L304

D301

G217E218

R299

K308Y229

G217E218

R299

K308Y229

a) b)

R299

K308

H302

R388

D394

E303

K279

L304

D301

G217E218

R299

K308Y229

a) b)

R299

K308

H302

R388

D394

E303

K279

L304

D301

G217E218

R299

K308Y229

G217E218

R299

K308Y229

a) b)

R299

K308

H302

R388

D394

E303

K279

L304

D301

Domain-domain Communication for tRNA Aminoacylation: Importance of Evolutionarily Conserved and Energetically Coupled Residues

Brianne Shane, Kristina Weimer, and Sanchita HatiDepartment of Chemistry, University of Wisconsin-Eau Claire, Eau Claire WI 54702

Acknowledgements: Research Corporation Cottrell College Science Award UWEC-Office of Research and Sponsored Programs

a) b) c)

Selective residues in the editing and catalytic domains of E. coli ProRS showing moderate to strong coupling

Evolutionarily Coupled Residues in E. coli ProRS

F359

L304

H302

F147R193

H208

Q211

F359

L304

H302

F147R193

H208

Q211

0.60.50.70.6

25242725

F147R193H208Q211

L304

0.81.01.00.8

27252626

F147R193H208Q211

H302

44414242

Distance(Å)

F359

Residues inediting domain

0.80.40.70.8

F147R193H208Q211

Coupling energy(kT*)

Residues incatalyticdomain

0.60.50.70.6

25242725

F147R193H208Q211

L304

0.81.01.00.8

27252626

F147R193H208Q211

H302

44414242

Distance(Å)

F359

Residues inediting domain

0.80.40.70.8

F147R193H208Q211

Coupling energy(kT*)

Residues incatalyticdomain

Coevolved residues obtained from the SCA of the ProRS family and their mapping on the 3D model structure of E. coli ProRS. a) The color scale linearly maps the data from 0 kT* (blue) to 1 kT* (red); b) The statistical coupling matrix where rows represent positions (N to C terminus, top to bottom) and columns represent perturbations (N to C terminus, left to right); c) Coupled residues obtained in b) are mapped on the E. coli ProRS 3D model structure. Residues selected for mutational studies are labeled.

SCA is based upon the assumption that “coupling of two sites in a protein, whether for structural or functional reasons, should cause those two positions to co-evolve” (5). The overall evolutionarily conservation parameter at a position i in the sequence of the chosen protein family is calculated and expressed as

where kT* is an arbitrary energy unit, Pi

x is the probability of any amino acid x at site i, and PMSA

x is the probability of x in the MSA. The coupling of site i with site j is calculated and expressed as

where Pix |j is the probability of x at site i dependent on perturbation at site j. We performed SCA on an alignment of 494 protein sequences of the ProRS family.

The SCA was performed by systematically perturbing each position where a specific amino acid was present in at least 50% of the sequences in the alignment. The initial clustering resulted in a matrix with 570 (residue number) 146 (perturbation site) matrix elements representing the coupling between residues. The SCA on the ProRS family demonstrates a group of residues which have coevolved in E. coli ProRS.

(5 Lockless, S. W., and Ranganathan, R. (1999) Science 286, 295-299.)

2stat ])[ln(* x

xMSA

xii PPkTG

2, )]/ln()[ln(* x

MSAxi

xj

xMSAj

xi

statji PPPPkTG

Statistical Coupling Analysis (SCA)

To explore the molecular basis of the long-range communication between functional and structural elements of E. coli prolyl-tRNA synthetase and probe the hypothesis that networks of interactions among evolutionarily conserved and energetically coupled residues are involved in the transmission of a signal from one functional site to the other. Statistical coupling analysis and site-directed mutagenesis have been employed to identify the communication network.

Objectives

Overexpression and Purification of Histidine-tagged E. coli ProRS Mutant Using Co2+-chelated Talon Resin

12% SDS PAGE gel pictures. a) Overexpressed E218A mutant after 0,1,2, and 4 hours of induction; b) Imidazole (10 -200mM) elution fractions; c) wild-type ProRS and E218A mutant (after concentrating the 100 and 150 mM imidazole elution fractions). M: Protein standard, FT: flow-through, W: wash. BioRad protein Assay: concentration of wild-type ProRS = 160.4 mg/ml and E218A mutant = 62.3 mg/ml.

a) b) c) M 0 1h 2h 4 h M FT W 10 25 50 100 150 200 WT 100 150

63.7 kD

78.0 kD45.7 kD

Our future work involves the continuation of the mutational studies to evaluate the impact of mutation (of key networking residues) on enzymatic functions. This will include the determination of kinetic parameters for aminoacylation, amino acid activation, and editing reactions for all the key mutants.

Future Work

• SCA study demonstrates that residues that are either evolutionarily conserved or coevolved constitute a distinguished set of interaction networks that are sparsely distributed in the domain interfaces. Residues of these networking clusters are within the van der Waals contact and appear to be the prime mediators of long-range communications between various functional sites located at different domains.

• Mutation of a single residue (E218 to alanine) has a drastic effect on the enzyme function, it affects the amino acid discrimination by E. coli ProRS. This study demonstrates that the mutation of the highly conserved E218 residue disrupted the interactions network between the editing and the catalytic domain.

Conclusions

Wild-type E. coli ProRS Exhibits Pre-transfer Editing Activity Against Alanine

6 Beuning and K. Musier-Forsyth (2000) PNAS V97, p. 8916-89207 Lloyd, A. J., Thomann, H. U., Ibba, M., and Soll, D. (1995) Nucleic Acids Res 23, 2886-2892.

E + ALA + ATP E.ALA~AMP+ PPi E + ALA + AMP + 2PiPPiase

a) Radioactive Assay (6) b) Spectroscopic Assay (7)

N

NHN

N

CH3S

NH2

O

N

NN

N

CH3S

NH2

HOCH2

HO OH

-

+

HOCH2

HO OH

OO P O

O

O-

-

Purine ribonucleosidephosphorylase

Ribose-1-phosphate2-amino-6-mercapto-7-methylpurine [Absmax=360nm]

Pi

2-amino-6-mercapto-7-methyl-purine ribonucleoside

N

NHN

N

CH3S

NH2

O

N

NN

N

CH3S

NH2

HOCH2

HO OH

-

+

O

N

NN

N

CH3S

NH2

HOCH2

HO OH

-

+

HOCH2

HO OH

OO P O

O

O-

-HOCH2

HO OH

OO P O

O

O-

-

Purine ribonucleosidephosphorylase

Ribose-1-phosphate2-amino-6-mercapto-7-methylpurine [Absmax=360nm]

Pi

2-amino-6-mercapto-7-methyl-purine ribonucleoside

Time (min)

PP

ire

leas

ed (

nm

ol)

0 10 20 30 40-1

0

1

2

3

4

5

Time (min)

PP

ire

leas

ed (

nm

ol)

0 10 20 30 40-1

0

1

2

3

4

5

32P-ATP

Pro-AMP

Ala-AMP Ala+ AMP

32PPi32P-ATP

Pro-AMP

Ala-AMP Ala+ AMP

32PPi

Mutation of E218 Has Significant Effect on Substrate Specificity and Binding

Pyrophosphate Assay

y = 0.0122x + 0.0168

y = 0.0255x - 0.0289

0

0.2

0.4

0.6

0.8

0 20 40 60 80Pyrophosphate (nmol)

Abso

rban

ce (3

60nm

)

b)a)

Na2P2O7

KH2PO4

Pre-transfer editing reaction

0

0.2

0.4

0.6

0.8

0 10 20 30 40Time (min)

Abso

rban

ce (3

60 n

m)

Pro (WT)Ala (WT)Pro (E218A)Ala (218A)

Editing of Errors in Selection of Amino Acids for Protein Synthesis: Pre- and Post-transfer Editing Pathways (2)

tRNAE·AA E·AA-AMP E·AA·tRNA

E + AA-tRNA

ATP PPi AMP

Amino Acid (AA)

E + AA + AMP+ tRNA

E·AA-AMP·tRNA

+ tRNA

Pre-transfer editing

E + AA + tRNA

Post-transfer editing

E + AA + AMP

- tRNA

(2 Jakubowski, H., and Goldman, E. (1992), Microbiol. Rev. 56, 412-429.)

Pyrophosphate assay to examine the catalytic efficiency of mutant protein. a) Comparison of standard curves using Na2P2O7 and KH2PO4 as the source of phosphate; b) The pre-transfer editing reaction with wild-type and E218A mutant carried out at room temperature using 2 µM enzyme, 3 mM ATP, 100 mM proline or 500 mM alanine.