Download Poster - Center for BioMolecular Modeling

Myosin: Mighty Morphing Movement Molecule
Cudahy SMART Team: George Ademi, Sal Munoz, Rachel Dombrowski, Samantha Brzezinski,
Paige Broeckel, , Jason Hauk, Katya Tolbert, Katharine McDonald, CeeCee Schoemann
Advisors: Dan Koslakiewicz and Dean Billo
Mentor: Thomas Eddinger, Ph.D., Biological Sciences, Marquette University
Abstract
II. Structural Organization of Myosin Heavy Chain
Muscle contraction is caused by the contractile protein myosin, which exists
in various isoforms (SMA and SMB) in different muscle types such as smooth,
cardiac, and skeletal. Current data suggests smooth muscle myosin is in a
non-functional conformation state until light chain 20, associated with the
myosin head, is phosphorylated. This allows either head to freely bind to
actin, a protein also involved in contraction. The contractile process is
initiated with ATP hydrolysis in the ATP binding region. The release of the
products from ATP hydrolysis causes the “lever arm” portion of each myosin
head to bend relative to the “motor domain,” pulling the actin fibers closer
together, shortening the muscle cells for movement. If the contractile process
is disrupted, bodily function is impaired. These key areas were modeled by
the Cudahy SMART (Students Modeling A Research Topic) Team using 3D
printing technology. Aortic coarctation, a developmental problem, involves a
constriction of the proximal aorta, increasing blood pressure by narrowing
the aorta and changing vascular smooth muscle in this region of vessel. While
surgery can correct the constriction and prolong life, there remain long-term
consequences, reducing life span. Drug treatments for a specific smooth
muscle problem can be complicated, altering functioning of other smooth
muscles, causing undesired side effects such as incontinence. The question
researchers face is targeting of drugs to a specific smooth muscle tissue.
Further understanding of smooth muscle function and regulation helps to
better treat, prevent, and or cure this and other smooth muscle diseases.
Myosin is the major contractile protein in muscle cells. Experiments have
been performed to determine the structure of myosin (Figure 1: Myosin
Segmentaion). By identifying and understanding the structure of myosin, a
clearer picture of how it functions emerges. The myosin head is the
“business” end of the molecule. It hydrolyzes ATP and interacts with actin to
generate force and cause shortening.
I. Types of Muscle
Movement of the body, including internal organs and blood, is accomplished
through the action of muscles. Muscle cells come in 3 varieties: skeletal,
smooth, and cardiac.
Smooth
Smooth muscle is located in many areas of the body, including blood vessels,
the digestive tract, and the bladder. Smooth muscle cells are not striated.
Contraction still occurs and results in a shortening of cells.
Muscle movement cannot be accomplished without the interaction between myosin and actin filaments. The three types of
muscle all contract using actin and myosin, however, smooth muscle myosin utilizes a different mechanism to facilitate
interaction between actin and myosin. This interaction results from a variety of chemical changes. Stimulation of smooth
muscle causes increased intracellular Ca2+, which activates a myosin light chain kinase (MLCK) that phosphorylates myosin
light chain (MLC20) and allows myosin binding to actin and cross-bridge cycling as shown below.
• Mornet, et al. used several different
proteases to cleave the myosin head
• Results produced similar sized fragments
• Dashed lines indicated that cleavage
occurred in almost same range resulting
in approximate sizes 25kD, 50kD, 20kD
• Trypsin (final line) was the only area to
produce different sized fragments
• Concluded there are 3 different domains
in myosin heavy chain
Figure 1: Myosin Segmentation
III. Identifying Structural/Functional Domains
Each domain contains segments responsible for the overall function of
myosin. The 25kD fragment (green) includes the ATP binding site (lime
green), responsible for ATP hydrolysis. The 50kD fragment (purple) is the
largest and most distal end and includes the actin binding sites (magenta
and dark orchid) on either side of the binding cleft. The last COOH 20kD
fragment (yellow) includes some amino acids that are close to the ATP
binding site, the convertor domain (red), and the lever arm (blue).
PDB File: 1BR1
PDB File: 1BR1
A
(A) Myosin heads in non-conformational state
(B) MLCK phosphorylates myosin light chain 20
and myosin heads separate
(C) ADP and Pi are present in the binding domain,
myosin heads bind to actin chain
(D) ADP and Pi are released, the power stroke is
initiated causing the lever arm to move the
myosin head
(E) Myosin head remains bound to the actin
following the power stroke
(F) ATP enters the binding domain on myosin
(G) The ATP allows it to myosin to be released
from actin
ATP hydrolysis causes lever arm to return to 450
for another cycle
Relaxed Smooth Muscle Cell
Skeletal
Skeletal muscle is found attached to bones. Their primary purpose is to move
the skeleton through contractions of the muscle cells. Structurally, skeletal
muscle is striated (alternating dark and light bands) due to the organization
of the thick and thin filaments. Contraction changes the relationship of these
filaments, changing the striation pattern and causing shortening.
Cardiac
Cardiac muscle is located in the heart. It is responsible for causing the heart
beat. Cardiac muscle cells are also striated. These cells contract in unison
when they are in direct contact, generating power to pump blood.
V. Proposed Action of Smooth Muscle Myosin in Movement
C
G
D
F
E
N
Contracted Smooth Muscle Cell
Eddinger, T., Meer, D. 2001
Front
Back
IV. Smooth Muscle Myosin Isoforms – SMA and SMB
VI. Conclusions
Myosin exists in almost every cell type and in various forms. Data (Figures 2, 3, 4) help identify unique functional properties
of two of the major myosin isoforms, SMA and SMB. Structurally, SMB has 7 additional residues in the ATP binding domain.
It correlates with increased ATP hydrolysis, and an increased contractile velocity. With faster shortening of the cells, force
generation can occur faster, allowing a faster response.
Smooth muscle surrounds all hollow organs in the body, including blood
vessels, airways, digestive, urinary and reproductive tracts. By
understanding the processes and functions of smooth muscle myosin,
scientists may be able to grasp its role in smooth muscle diseases. For
example aortic coarctation (CoA) is identified by a narrowing of the
proximal aorta, causing an increase in blood pressure and associated with
changes in the vascular smooth muscle cells in this region of vessel. This
condition is generally diagnosed early following birth. Surgery can correct
the anatomical narrowing, but long-term consequences that reduce overall
wellness of the affected individual remain. Hypertension (high blood
pressure) and ‘re-coarcting’ of the aorta are two potential outcomes that
may occur later in life. Irreversible changes in smooth muscle cell function
and protein expression (including SM myosin isoforms) may be responsible
for the long term consequences of CoA following surgical correction. These
changes may also be involved in other developmental diseases and
pathological conditions. Pharmacological treatments for a specific smooth
muscle condition pose a problem as these drugs may also alter function of
other smooth muscle tissues. This inability to target specific tissues may
cause undesired side effects. Researchers face the task of finding drugs that
act solely on a specific smooth muscle tissue.
Though drastically different in appearance, all muscle cells contract to shorter
lengths. The presence of the contractile protein myosin is responsible for the
shortening of muscle cells. Using a second protein, actin, myosin generates a
power stroke capable of contracting a cell.
Figure 2: Regions of the Stomach
http://apbrwww5.apsu.edu/thompsonj/Anatomy%20&%20Physiology/2010/2010%20Exam%20Reviews/Exam%201%20Review/Ch04%20Muscle%20and%20Nervous%20Tissues.htm
B
• 3 main regions (Fundus, Body, and
Antrum) are divided into 10
smaller sections
• SMA and SMB content of each
section
was
analyzed
for
abundance
Figure 3: Stomach Region Isoform Composition
• Sections 1-4: SMB is less than 20 %
• Sections 5-7: SMB is about 50 %
• Sections 8-10: SMB is 80 % or more
of the myosin in the stomach
Figure 4: Isoform Composition Contractile Velocity
• Velocity of contraction of fundic
cells, sections 1-4 , contract at a
lower rate
• Velocity of contraction of the antral
cells, section 8-10, contract at a
very high rate
References
Chinthalapudi, K. Heissler, S. M., Manstein, D. J. (2012). Crystal structure of human non muscle myosin 2C in pre-power stroke state. Protein Data Bank Retrieved on 20 January 2014: http://www.rcsb.org/pdb/explore.do?structureId=2YCU This data was used in the program Chimera by D.R. Swartz(Delaware Valley College) to determine ATP binding orientation.
Garland Science. (2009, April 21). Myosin. Retrieved February 5, 2014 from https://www.youtube.com/watch?v=j8F5GGPACkQ
Dominguez, R., Freyzon, Y., Trybus, K., Cohen, C. (1998). Crystal Structure of a Vertebrate Smooth Muscle Myosin Motor Domain and Its Complex with the Essential Light Chain: Visualization of the Pre-Power Stroke State. Cell. 94: 559-571.
Mornet, D., Ue, K., Morales, M. (1984). Proteolysis and the domain organization of myosin subfragment 1. Proceedings of the National Academy of Sciences USA 81:736-739.
Eddinger, T., Meer, D. (2001). Single rabbit stomach smooth muscle cell myosin heavy chain SMB expression and shortening velocity. American Physiological Society 280: C309-C316.
Woodhead, J., Zhao, F., Craig, R., Egelman, E., Alamo, L., Padron, R. (2005). Atomic model of a myosin filament in the relaxed state. Nature 436/25: 1195-1199.
SMART Teams are supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number
8UL1TR000055. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH