Investigating Novel Molecular Events Involved in Ca2+-

Calmodulin Activation of Smooth Muscle Myosin Light Chain

Kinase Using Hydrogen-Deuterium Exchange Mass
Spectrometry

By Lily Choi

Dr. Michael Walshs Lab

Co-supervised by Dr. John Chik
 Smooth Muscle Contraction

Ca2+ Ca2+ Channel Receptor

PIP
2
GEF RhoA-
PLC GTP
Ca2+
IP ZIP
K RO
3 Relaxatio ILK K
n
ATP Pi

ML MLC CPI-
CK Myosin P P 17
Ca2+ AD H2 0
P + actin
PK
ActomyosinP C

ATPcontractio AD
n P
 Objective

Identify critical molecular events involved in

Ca2+-Calmodulin activation of Myosin Light
Chain Kinase using Soybean Calmodulin
Isoforms (SCaM) and Hydrogen Deuterium
Exchange Mass Spectrometry
 Myosin Light Chain Kinase (MLCK) and
1 MLCK Calmodulin
526
(CaM) 776 815 972
Catalytic domain

ATP binding site Regulatory

Actin binding
domain domain
Autoinhibitory
domain CaM-binding domain
CaM

Apo-CaM Ca2+-CaM CaM-MLCK Peptide Comple

Van Lierop et al. (2002) . Journal of Biological Chemistry 277(8): 6550-6558c
A Proposed Mechanism of Ca2+-CaM Activation
of MLCK

N
MLCK active site

Autoinhibitory domain +CaM-binding

Adomain
utoinh
ib
domain itory domain C
+CaM-b
inding

C
Soybean Calmodulin Isoforms (SCaM) A Unique
Opportunity to Investigate Ca2+-CaM Activation of
MLCK
Sequence 1 Sequence 2 Identity Score (%)
SCaM 1 SCaM 4 79
SCaM 1 hCaM 90
SCaM 4 hCaM 76

SCaM 4 SCaM
1

MLCK SCaM-4 is a
competitive
MLC
inhibitor of
SCaM-1 (both
K
bind the same
MLCK site) and
acts as a SCaM-1
mutant.

Inactive MLCK Active MLCK

 Hypothesis
Conformational differences between SCaM-1/MLCK and SCaM-4/MLCK
will reflect novel molecular events critical for calcium-calmodulin
mediated MLCK activation, such as changes in the autoinhibitory
domain of MLCK or the newly formed surfaces on CaM upon MLCK
1 526
binding 776 815 972
Catalytic domain

Actin binding domain ATP binding site Regulatory

domain

Autoinhibitory domain
CaM-binding domain

CaM-MLCK Peptide Complex

Van Lierop et al. (2002) . Journal of Biological Chemistry 277(8): 6550-6558c

 Hydrogen and Deuterium Exchange Mass
Spectrometry (HDX MS)
1. Protein labeling with deuterium 2. Protein digestion with proteases
or water over various time points (eg. Pepsin)
(SCaM and MLCK)
D
D

D D
R1 H O D
DD D
C N C D D
N C C D

H O R2 D D
D
D D

5. Data 4. QTRAP 3. Ultimate 3000

Collection MS HPLC
 X! Tandem in Trans-Proteomic Pipeline
Assigns the best sequence model for a given MS/MS spectrum of a
particular peptide.
A Comparison of Apo-SCaM-1 and Ca2+-SCaM-1
Deuterium Incorporation
An Example of a peptide that showed no change in
deuterium incorporation upon calcium binding to SCaM-1.

Deuterated Calcium-SCaM-1 at 0.5 minutes

Deuterated Calcium-SCaM-1 at 1 minute
A Comparison of Apo-SCaM-1 and Ca2+-SCaM-1
Deuterium Incorporation Over Time
An example of a peptide that shows a decrease in
deuterium incorporation at a calcium binding site upon
calcium binding to SCaM-1.

Deuterated Apo-SCaM-1 at 0.5 minutes

Deuterated Apo-SCaM-1 at 1 minute
 HDX MS Analysis
H O
Buried regions of a
protein, areas involved in
hydrogen bonding, or a
No
combination of both.
Exchange
H Areas of a protein
that are exposed
to the solvent.
Detect Protein
H Exchange
conformational
changes at the H
binding site due H
Detect
to a binding D conformational
event. D
D
H D changes due to a
D
H binding event in
D regions distal to
the binding site.

Detect no changes in deuterium

before and after a binding event-an
area relatively unaffected by the
event
Peptide Changes and Regions in SCaM-1

>1: increase in
deuteration

=1: No change in
deuteration

<1: Decrease in
deuteration
3D Structure of Peptide Changes in SCaM-1
H2 H2

H1 H1

H3
H3 H4
Red : decrease in
H4 deuteration

Blue: Increase in
deuteration

H5 H5
H8

H6
H8
H7
H6
H7

Apo-SCaM-1 Ca2+-SCaM-1
 Deuterated SCaM Coverage

Lower SCaM-4 coverage than SCaM-1 because the purified protein

 Undeuterated MLCK Coverage

Lower coverage due to the large size of MLCK and the low concentration of
protein that can be purified.
However, the areas of interest including the autoinhibitory (underlined) and
CaM binding domains (Italics) are consistently present in coverage data.
Can still use intact MLCK to gain information on the mechanism of
Summary

Good coverage of Undeuterated SCaMs and sufficient

coverage of MLCK.

A general decrease in deuteration as calcium binds SCaM-

1, particularly in the helices of EF hand motifs, as well as
peptides containing residues involved in calcium
coordination.
Future Directions

Collect more data for both SCaM-1 and SCaM-4 at different

time points

Purify SCaM-4 to a higher concentration to obtain better

deuterated coverage

Conduct binding experiments of SCaMs with intact MLCK

and a peptide of MLCK consisting of the autoinhibitory and
CaM binding domains.
Acknowledgements
Funding
Supervisor
Dr. Michael P. Walsh Kim Kertland
Co-supervisor
Dr. John Chik

Committee Members
Dr. Hans Vogel (SCaM provider)
Dr. Justin MacDonald (HPLC and Mass
Spectrometer Provider)

Lab Members
Cindy Sutherland (Protein Purification)
Lori Moffat
Dr. Iris Kathol
Dr. Yana Anfinogenova
Dr. Yu Gui
Dr. Kosuke Takeya
Dr. Jingti Deng

Summer Student
Anne Yang
Summary of Results See a general decrease in deuteration as calcium
binds SCaM-1, particularly in the helices of EF hand
motifs, as well as peptides containing residues
involved in calcium coordination.

Some unexpected results are that there was an

increase in deuteration in the EVDADGNGTIDFPEFL
peptide, a region for calcium binding in EF2.

EF hands showed varying degrees in deuteration

change. EF4>EF3>EF2 (increase) trend. Perhaps EF2
is a possible MLCK binding region- in proximity of
residues in mutational studies
(K30D, G40D, M36I).

Loop region MRSLGQNPTEA connecting EF1 and EF2

motifs
showed no change in deuteration-area relatively
unaffected by binding of calcium

Helix 3 showed the most decrease in deuterium

compared to all other helices.

Helix 1 and 8 at the very end of the N and C

terminals showed relatively small decreases in
deuterium change.

SCaM-4 showed decrease s in deuteration in several

helical areas, but to a greater extent than SCaM-1.
Closer conformation upon than SCaM-1 in the C-lobe-
consistent with NMR data and x-ray crystallography
Up Next..

Concentrate SCaM-4 to obtain more peptides that can

be compared to SCaM-1. An alternative is to re-purify
SCaM-4 to obtain a higher concentration.

Decrease the peak intensity cutoff in data collection to

look at peptides that may have been missed in both
SCaM-1 and SCaM-4

Decrease the volume of quenching solution by using a

higher concentration to reduce the dilution of proteins
during HDXMS

MLCK coverage is too low-purchased a peptide

containing the autoinhibitory AND CaM binding site. Try
binding experiments with SCaM-1 and SCaM-4 with this
peptide.
Basis of Project

Difference in MLCK Activation Ability

bCaM
SCaM-1
SCaM-4
SCaM-4111

K30E/M36I/G40D

Van Lierop et al. (2002)

Activation of Smooth
Muscle Myosin Light Chain
Kinase by Calmodulin .
Journal of Biological
Chemistry 277(8): 6550-
6558

SCaM 1 activates MLCK in a similar manner to bCaM

while SCaM 4 does not.
Basis of Project

Competitive Inhibition
1.8nM of bCaM

1.7nM of
SCaM-1
1.8nM of SCaM-
1 Van Lierop et al.
(2002) Activation
of Smooth Muscle
Myosin Light Chain
Kinase by
Calmodulin .
Journal of
Biological
Chemistry 277(8):
6550-6558

SCaM 4 is a competitive Inhibitor of SCaM 1

Strongly suggests that both isoforms bind the
same site on MLCK
Compare the Same Peptides Between Different Trials
Using Retention Times

A.AELRHVMTNL.G

Undeuterated Peptide Retention Time-8.04

Deuterated Peptide Retention Time-8.036

#D
Incorporated #D= Average Mass
Average Mass
 DNase I Binding to G-acting
decrease in actin deuteration
resulting from DNase binding

DNase I DNase I

G-actin
G-actin

increase in deuteration resulting

from DNase binding!

Consistent with trypsin cleavage data!

Analyst-Data Collection
Results
3D Structure of Peptide Changes in SCaM-1
H2 and SCaM-4
H2
H1
H1

H3
H4 H3
H4
Red> Purple > Pink :
decreases in deuteration
Ca2+-SCaM-1 Ca2+-SCaM-4
Blue: Increase in
deuteration
H5
Yellow: No change in H5
H8
deuteration H8

H7 H6
H7 H6
Smooth Muscle Contraction

Ca2+ Channel Receptor

Ca2+

PIP
2
PLC
Ca2+
IP
3 Relaxati
on
ATP Pi

ML MLC
CK Myosin P P
Ca2+ AD H2 0
P + actin

Actomyosin
P

ATPcontractioAD
n P