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Oct 4, 2013 (3 years and 10 months ago)

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Aminoacyl tRNA Synthetases in Translation

Aminoacylation of tRNA is a Two
-
step Reaction

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
-
tRNA
Pro
.
Editing domain of bacterial ProRSs selectively hydrolyzes alanyl
-
tRNA
Pro
(
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.)

Abstract

Aminoacyl

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

interdomain


communications 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.

Domain
-
domain Communication for
tRNA

Aminoacylation
: Importance of Evolutionarily Conserved and
Energetically Coupled Residues


Brianne Shane
,
Kristina Weimer
, and
Sanchita

Hati

Department 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

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,
P
i
x

is the probability of any amino acid
x

at site
i
, and
P
MSA
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.)

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

Co
2+
-
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 kD

45.7 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
-
8920

7
Lloyd, A. J.,
Thomann
, H. U.,
Ibba
, M., and
Soll
, D. (1995)
Nucleic
Acids Res

23
, 2886
-
2892.


a) Radioactive Assay (
6
)





b) Spectroscopic Assay (
7
)

Mutation of E218 Has Significant Effect on Substrate Specificity and Binding


b)

a)

Na
2
P
2
O
7

KH
2
PO
4

Editing of Errors in Selection of Amino Acids for Protein Synthesis:

Pre
-

and

Post
-
transfer Editing Pathways (
2
)


(
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
Na
2
P
2
O
7

and KH
2
PO
4

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
.