Transcript MUSCLE AND MOLECULAR MOTORS Medical Biochemistry, Lecture 29 Lecture 29, Outline • Muscle proteins and structure • Protein interactions involved with muscle contraction • Structure of.

MUSCLE AND MOLECULAR
MOTORS
Medical Biochemistry, Lecture 29
Lecture 29, Outline
• Muscle proteins and structure
• Protein interactions involved with muscle
contraction
• Structure of tubulin
• Microtubules and cilia
Thick and Thin Filaments
Thick Filaments – Myosin
• Composed of six highly conserved polypeptide
chains
• Two 220 kDa heavy chains that have elongated
globlular heads, and long fibrous helical tails.
Each head region contains an ATP-binding
site/ATPase function
• Two pairs of light chains, termed essential light
chains and regulatory light chains. These
interact with the globular heads of the heavy
chains, and regulate ATPase functions
depending on their phosphorylation status.
MYOSIN STRUCTURE
ATPase
Conserved Hydrophobic residues
at positions a and d in the helices
Structure of Myosin Head Region
Thin Filaments - Actin
• Exists as a monomer, termed G-Actin, at low
ionic strength and can bind one ATP
• At physiological ionic strength (plus Mg2+), the
G-actin forms fibrous polymers termed F-actin.
ATP hydrolysis occurs during this process and
ADP remains bound to each F-actin subunit
• F-actin is the core of the thin filament, and each
monomer is capable of binding one myosin
globular head. The coil repeats at roughly every
seventh actin unit.
Actin Monomer Structure
Thin Filaments:
Troponin/Tropomyosin
• Tropomyosin: a two chain fibrous protein that
attach to F-actin in the groove between its
filaments
• Troponins: three protein components, troponin C,
a calcium binding protein like calmodulin;
troponin I, which blocks the myosin binding site
on F-actin; troponin T which binds to tropomyosin
and the other troponins
Steps in Muscle Contraction
• In the contraction cycle, myosin and actin are
already bound in a “low energy” state
• Step 1. ATP binds to myosin, causing a
conformational shift in myosin and dissociation of
it from actin
• Step 2. Myosin bound ATP is rapidly hydrolyzed
to form a stable “high energy” myosin-ADP-Pi
complex
• Step 3. Actin binds to the myosin-ADP -Pi
complex. (This would usually occur in response to
nerve/Ca2+ stimulation)
Steps in Muscle Contraction (cont.)
• Step 4. The myosin-ADP-Pi complex
sequentially releases Pi , followed by
release of ADP (this process is termed the
power stroke). The accompanying large
conformational change in the myosin head
pulls actin about 10 nm toward the center of
the sarcomere.
• Myosin remains bounds to actin in the “low
energy” state until another ATP binds to
reinitiate the contraction cycle
The Role of Calcium and
Troponins in Contraction
• The calcium released from the sarcoplasmic
reticulum will bind to troponin C (like
calmodulin, 4 Ca/molecule). The
conformational change in TpC leads to
adjustments in the conformations of TpI,
TpT and tropomyosin. The net effect of
these conformational shifts is the movement
of TpI to expose the myosin binding cleft on
the actin.
Other protein
components of
muscle fibrils
Other Proteins in Thick and Thin
Filaments
Titin (also called connectin)
Largest protein in the body,
33,000 amino acids, 3600 kDa in
size; it surrounds the thick
filament and may be involved in
controlling filament length and
relative flexibility
Nebulin also v. large, 7000 amino
acids, it traverses the actin thin
filaments and may be involved in
controlling thin filament length
a-Actinin anchors the thin filament
to the Z-line
Smooth Muscle Contraction
• Smooth muscle thin filaments contain no tropinins, only
actin and tropomyosin
• Contraction is still calcium dependent though, because of
calcium/calmodulin activation of myosin light chain kinase
(MLCK), which phosphorylates the regulatory light chain of
myosin, stimulating actin binding/movement. A specific
phosphatase reverses this.
• Activity of MCLK can be negatively regulated hormonally
by epinephrine, and activated by oxytocin (pitocin). The
asthma medication, albuterol, acts to inhibit MLCK activity
Muscular Dystrophy
(also see case report in Ch. 65, pp 857-859
• Duchenne’s muscular dystrophy is a fatal muscle wasting
disease with onset at ages 2-5 and progressive muscle loss.
Patients usually die of infection or respiratory problems by
age 30
• Defect is caused by complete absence of dystrophin, a 427
kDa protein that constitutes only 0.01% of total muscle
protein.
• The structural motifs of dystrophin are shown on the next
slide. It forms antiparallel tetramers linked to actin on the
cytoplasmic face of muscle cell plasma membrane. Because
it is complexed with other surface glycoproteins, it likely
plays a membrane structural role and possibly mediates
signal transduction pathways
Structural homology of dystrophin
to other anchor-type proteins
Dystrophin
Microtubule Functions
• Intracellular microtubule network/vesicle
translocation
• Mitotic spindle
• Cilia and flagella
GTP dependent
polymerization
Axoneme structure
of cilia
Absence of dynein
results in Kartagener
Syndrome, characterized
by male sterility and
chronic respiratory
Infections due to nonfunctional cilia and
flagella
Ciliary Axoneme Structure (cont)
ATP-dependent Ciliary Movement
Microtubule-based Axonal Transport
Microtubule-Mediated Organelle
and Vesicular Transport
The hydrolysis of ATP by kinesin provides the energy
for transport of the vesicle or organelle along the
microtubule
Clinically Used
Microtubule
Inhibitory Drugs
Vinca alkaloids – vinblastine
and vincristine
Colchicine
Taxol (paclitaxel)
Microtubule Inhibitors
• Colchicine – isolated from the stems of the
autumn crocus and meadow saffron. It
inhibits tubulin polymerization, and used
clinically most often to treat the pain
associated with gout. Exact mechanism
unclear, but related to inhibition of white
cell inflammation responses to the uric acid
crystals in gout patients
Microtubule Inhibitors (cont)
• Vinca alkaloids – isolated from the Madagascar periwinkle,
these compounds inhibit tubulin polymerization. Used
clinically for some cancer chemotherapies
• Taxol – isolated from the Pacific yew tree, it acts by
stimulating and stabilizing microtubule polymerization. It is
fast becoming a standard chemotherapeutic for many types
of breast and ovarian cancers.
• NOTE: mechanistically it is thought that inhibition of
mitotic spindle formation is the primary effect of both
classes of inhibitors