Role of Histidine 55 in the
Dimerization of
the Cytoplasmic Dynein
Subunit LC8
Loren Cochrun
Dr. Elisar Barbar
Department of Biochemistry & Biophysics
Oregon State University
September 1, 2004
Intracellular Movement

“Motor proteins” = Large protein complexes responsible for
intracellular movement

Two types of microtubule associated motor proteins:
 Dynein = transport towards the center of the cell
 Kinesin = transport toward the cell periphery
Intracellular Movement



Heavy Chain (red) attaches to
microtubules
Protein Subunits (black) bind to
“cargo”
 Cargo = molecule being
transported within the cell
 Vesicle in a neuron
 Golgi vesicle
 Chromosomes during mitosis
Chemical energy is converted into
mechanical energy (hydrolysis of
ATP) which allows movement
along the microtubules
“Cargo”
Microtubule
Cytoplasmic Dyneins




Function in Cells
Positioning of organelles
Chromosome alignment
& segregation during cell
division
Transfer of materials
between ER and Golgi
complex
Viral transportation
Picture of a dynein using
electron microscope
(www.reporter.leeds.ac.uk/488/)
Cytoplasmic Dyneins
Composition

2 Heavy chains



2 Light Intermediate chains



ATP binding sites
Microtubule binding sites
Assembly of protein complex
Cargo binding
Other protein subunits


LC8 dimer
Can only function if all
protein subunits are present
LC8




10 kDa light chain
Dimer at physiological pH
LC8 has been observed to increase order in other
dynein subunits upon binding
Highly conserved (94% of the amino acid
sequence between Drosophila and human)


Suggests that it plays a critical role in the dynein
complex
Possible regulatory mechanism in dynein assembly
and/or cargo binding
Objective

To understand the mechanism by which the
dynein complex assembles and binds to its
cargo

To identify the amino acid residues responsible for
LC8 dimerization
Hypothesis

Histidine 55 is important for LC8 dimerization
Amino acid residues responsible
for LC8 dimerization

LC8 dimerization is pH dependent


Low pH  Monomer
High pH  Dimer

Charge on histidine is also pH dependent

Three histidine residues appear near dimer interface

Used pH titration & NMR (Nuclear Magnetic
Resonance) to examine the residues that affect
dimerization by monitoring their chemical shifts
3D NMR Spectrum
Carbon
chemical
shifts
Histidine
Residues
Proton chemical shifts
Proposed Histidine 55, 68, & 72
Peaks
68 Dimer
55 Monomer
68 Monomer
55 Dimer
72 Monomer
72 Dimer
Histidine 55

Histidine 55 is located directly at
the dimer interface

Low pH  Histidine becomes
protonated (positively charged)

Two positive charges on His 55
cause repulsion  makes the
LC8 dimer dissociate into two
monomers

LC8 monomers retain secondary
and tertiary structures 
suggesting regulatory role in
assembly
LC8
+His
+ Repulsion
His
His 55
LC8
His 55’
Chemical Shifts

13C Chemical Shifts
138.0
137.8
137.6
137.4
137.2
137.0
136.8
136.6
136.4
136.2
1H Chemical Shifts
Dimer
8.70
55M
68M
68D
Monomer
72M
72D
2
4
6
pH
8
Monomer
8.65
8.60
55D
ppm

When histidine 55 was replaced with alanine 
neutral charge  dimer (independent of pH)
When histidine 55 was replaced with lysine 
positively charged  monomer (independent of pH)
When histidine 55 was left unaltered…
ppm

55M
55D
8.55
8.50
68M
68D
8.45
8.40
Dimer
8.35
8.30
2
4
6
pH
8
72M
72D
Predictions for the 3D NMR
Spectrum

Monomer peaks decrease in intensity as pH increases

Dimer peaks increase in intensity as pH increases

Histidine residue that shifts the most is at the dimer
interface
Monomer
Monomer/Dimer
Monomer/Dimer
Dimer
Conclusions

His 55 affects dimerization of LC8

The ionization state of the interface histidine
residue is important in LC8 dimer assembly
Future Studies



Continue pH titration of
LC8 through pH 8
Repeat the experiments
with other LC8 mutants
to verify the histidine
peak assignments
Examine the interaction
of LC8 on other protein
subunits within the
dynein complex
Acknowledgements

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Elisar Barbar
Afua Nyarko
Kevin Ahern
Howard Hughes
Medical Institute