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 “Motor proteins” = Large protein complexes responsible for intracellular movement Two types of microtubule associated motor proteins: Two types of microtubule associated motor proteins: Dynein = transport towards the center of the cell Dynein = transport towards the center of the cell Kinesin = transport toward the cell periphery Kinesin = transport toward the cell periphery

Intracellular Movement Heavy Chain (red) attaches to microtubules Heavy Chain (red) attaches to microtubules Protein Subunits (black) bind to “cargo” Protein Subunits (black) bind to “cargo” Cargo = molecule being transported within the cell Cargo = molecule being transported within the cell Vesicle in a neuron Vesicle in a neuron Golgi vesicle Golgi vesicle Chromosomes during mitosis Chromosomes during mitosis Chemical energy is converted into mechanical energy (hydrolysis of ATP) which allows movement along the microtubules Chemical energy is converted into mechanical energy (hydrolysis of ATP) which allows movement along the microtubules Microtubule “Cargo”

Cytoplasmic Dyneins Function in Cells Function in Cells Positioning of organelles Positioning of organelles Chromosome alignment Chromosome alignment & segregation during cell division & segregation during cell division Transfer of materials between ER and Golgi complex Transfer of materials between ER and Golgi complex Viral transportation Viral transportation Picture of a dynein using electron microscope (

Cytoplasmic Dyneins Composition Composition 2 Heavy chains 2 Heavy chains ATP binding sites ATP binding sites Microtubule binding sites Microtubule binding sites 2 Light Intermediate chains 2 Light Intermediate chains Assembly of protein complex Assembly of protein complex Cargo binding Cargo binding Other protein subunits Other protein subunits LC8 dimer LC8 dimer Can only function if all protein subunits are present Can only function if all protein subunits are present

LC8 10 kDa light chain 10 kDa light chain Dimer at physiological pH Dimer at physiological pH LC8 has been observed to increase order in other dynein subunits upon binding 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) Highly conserved (94% of the amino acid sequence between Drosophila and human) Suggests that it plays a critical role in the dynein complex Suggests that it plays a critical role in the dynein complex Possible regulatory mechanism in dynein assembly and/or cargo binding 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 understand the mechanism by which the dynein complex assembles and binds to its cargo To identify the amino acid residues responsible for LC8 dimerization To identify the amino acid residues responsible for LC8 dimerization

Hypothesis Histidine 55 is important for LC8 dimerization Histidine 55 is important for LC8 dimerization

Amino acid residues responsible for LC8 dimerization LC8 dimerization is pH dependent LC8 dimerization is pH dependent Low pH  Monomer Low pH  Monomer High pH  Dimer High pH  Dimer Charge on histidine is also pH dependent Charge on histidine is also pH dependent Three histidine residues appear near dimer interface 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 Used pH titration & NMR (Nuclear Magnetic Resonance) to examine the residues that affect dimerization by monitoring their chemical shifts

3D NMR Spectrum 3D NMR Spectrum Proton chemical shifts Carbon chemical shifts Histidine Residues

Proposed Histidine 55, 68, & 72 Peaks 72 Dimer 55 Monomer 68 Monomer 72 Monomer 55 Dimer 68 Dimer

Histidine 55 Histidine 55 is located directly at the dimer interface Histidine 55 is located directly at the dimer interface Low pH  Histidine becomes protonated (positively charged) Low pH  Histidine becomes protonated (positively charged) Two positive charges on His 55 cause repulsion  makes the LC8 dimer dissociate into two monomers 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 monomers retain secondary and tertiary structures  suggesting regulatory role in assembly His + + His Repulsion LC8 His 55’ His 55

Chemical Shifts When histidine 55 was replaced with alanine  neutral charge  dimer (independent of pH) 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 replaced with lysine  positively charged  monomer (independent of pH) When histidine 55 was left unaltered… When histidine 55 was left unaltered… Monomer Dimer Monomer Dimer

Predictions for the 3D NMR Spectrum Monomer peaks decrease in intensity as pH increases Monomer peaks decrease in intensity as pH increases Dimer peaks increase in intensity as pH increases Dimer peaks increase in intensity as pH increases Histidine residue that shifts the most is at the dimer interface Histidine residue that shifts the most is at the dimer interface

Monomer Monomer/Dimer Dimer

Conclusions His 55 affects dimerization of LC8 His 55 affects dimerization of LC8 The ionization state of the interface histidine residue is important in LC8 dimer assembly The ionization state of the interface histidine residue is important in LC8 dimer assembly

Future Studies Continue pH titration of LC8 through pH 8 Continue pH titration of LC8 through pH 8 Repeat the experiments with other LC8 mutants to verify the histidine peak assignments 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 Examine the interaction of LC8 on other protein subunits within the dynein complex

Acknowledgements Elisar Barbar Elisar Barbar Afua Nyarko Afua Nyarko Kevin Ahern Kevin Ahern Howard Hughes Medical Institute Howard Hughes Medical Institute