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Domain-domain Communication for tRNA Aminoacylation: Importance of Evolutionarily Conserved and
Energetically Coupled Residues
Acknowledgements:
Research Corporation Cottrell College Science Award
UWEC-Office of Research and Sponsored Programs
Brianne Shane, Kristina Weimer, and Sanchita Hati
Department of Chemistry, University of Wisconsin-Eau Claire, Eau Claire WI 54702
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.)
Aminoacyl tRNA Synthetases in Translation
Evolutionarily Conserved or Coupled Residues Constitute a Sparse but Contiguous
Network of Interactions
Long-range Communications in Bacterial Prolyl-tRNA Synthetases
Binds amino
acid and
ATP to form
an activated
intermediate
known as
amino acid
adenylate
Editing domain
a)
Catalytic
domain
Transfer RNA (tRNA)
Aminoacyl-tRNA Synthetase (ARS)
Aminoacyl-tRNA
(AA-tRNA)
Growing Protein Chain
Binds specific
tRNA and
orients it
towards the
catalytic
domain
tRNA
binding
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).
Statistical Coupling Analysis (SCA)
(mRNA)
stat
Gi
N
O
C
O-
O
+H2N
-O
+
+
P
O
O-

P
O-

Proline
ProRS
O
O
O
P
N
O
O
N
N
O
-PPi
+H2N
NH 2
N
O
x
O
O-

OH
OH
ProRS•Pro-AMP
(Aminoacyl-adenylate)
ATP
Step 2. Amino acid is transferred to 3′-end of tRNA
5′
A76
O
NH 2
O
3′
N
O
OH
OH
+
+H2N
O
N
N
C O P O
O-
5′
O
A76
O
O
3′
N
O
 kT *  [ln( Pi  j
x
x
PPi
E·AA
- tRNA
E + AA + AMP
tRNA
b)
c)
F359
L304
R193
F147
x
2
/ PMSA )]
S. W., and Ranganathan, R. (1999) Science 286, 295-299.)
Residues in
editing
domain
H208
H302
L304
Q211
F359
E·AA-AMP·tRNA
E + AA + AMP+ tRNA
(2 Jakubowski, H., and Goldman, E. (1992), Microbiol. Rev. 56, 412-429.)
1h
2h
b) M
4h
FT
W 10
25
c)
50 100 150 200
WT
100
150
63.7 kD
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.
E + ALA + ATP  E.ALA~AMP+ PPi
a) Radioactive Assay (6)
PPiase
E + ALA + AMP + 2Pi
b) Spectroscopic Assay (7)
CH3
Ala-AMP
4
+
Ala+ AMP
3
N
P-ATP
PPi
2
N
Pi
NH2
HOCH2
32
32
-
S
N
N
O
HO OH
1
Purine ribonucleoside
phosphorylase
2-amino-6-mercapto7-methyl-purine
ribonucleoside
Pro-AMP
0
-1
0
10
20
30
Residues in
catalytic
domain
F147
R193
H208
Q211
F147
R193
H208
Q211
F147
R193
H208
Q211
Distance
(Å)
27
25
26
26
25
24
27
25
44
41
42
42
Coupling
energy
(kT*)
0.8
1.0
1.0
0.8
0.6
0.5
0.7
0.6
0.8
0.4
0.7
0.8
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.
S
CH3
40
O
N
6
O-
P
-
O
NH
N
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.
HOCH2
O
N
Time (min)
NH2
2-amino-6-mercapto-7-methyl
purine [Absmax=360nm]
HO OH
Ribose-1-phosphate
Mutation of E218 Has Significant Effect on Substrate Specificity and Binding
a)
b)
Pyrophosphate Assay
Pre-transfer editing reaction
0.8
y = 0.0255x - 0.0289
Na2P2O7
0.6
E + AA + tRNA
Post-transfer editing
K308
78.0 kD
45.7 kD
Selective residues in the editing and catalytic
domains of E. coli ProRS showing moderate to
strong coupling
E·AA·tRNA
+ tRNA
Pre-transfer editing
x
x
PMSA  j )  ln( Pi
H302
AMP
E·AA-AMP
0
Evolutionarily Coupled Residues in E. coli ProRS
Editing of Errors in Selection of Amino Acids for Protein Synthesis: Preand Post-transfer Editing Pathways (2)
E + AA-tRNA
D394
0.8
a)
L304
R388
5
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.
Pro-tRNAPro
Amino Acid (AA)
H302
E303
O
+H2N
ATP
M
OH
O C
tRNAPro
a)
x
2
PMSA )]
(5 Lockless,
-AMP
OH OH
stat
Gi, j
N
OH OH
x
where kT* is an arbitrary energy unit, Pix is the probability of any amino acid x at site i, and
PMSAx is the probability of x in the MSA. The coupling of site i with site j is calculated and
expressed as
N
N
C O P O
O-
H302
E303
Wild-type E. coli ProRS Exhibits Pre-transfer Editing Activity Against Alanine
 kT *  [ln( Pi
Aminoacylation of tRNA is a Two-step Reaction
Step 1. Activation of the amino acid.
ARS activates an amino acid in the presence of ATP to form the aminoacyl-adenylate
intermediate:
NH 2
R299
K279
Overexpression and Purification of Histidine-tagged E. coli ProRS Mutant Using
Co2+-chelated Talon Resin
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
(EF)
Ribosome
Messenger RNA
R299
K279
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.
GTP
Elongation factor
D301
K308
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.
ATP
D301
D394
Absorbance (360nm)
+
b)
R388
Objectives
Amino Acid
b)
K308
Y229
L304
Proofreading reaction
to remove noncognate amino acid
attached to tRNA
PPi released (nmol)
3'
K308
Y229
R299
R299
E218
E218
G217
G217
(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 MusierForsyth, K. (2002), Biochemistry 41, 7108-7115.)
5'
a)
Absorbance (360 nm)
Abstract
y = 0.0122x + 0.0168
KH2PO4
0.4
0.2
0.6
Pro (WT)
Ala (WT)
Pro (E218A)
Ala (218A)
0.4
0.2
0
0
0
20
40
60
Pyrophosphate (nmol)
80
0
10
20
30
40
Time (min)
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.
Conclusions
•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.
Future Work
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.