BIOC 212 Lecture Notes - Lecture 3: Apoptosis, Proteasome, Organelle
28 May 2018
School
Department
Course
Professor
Protein Folding in the Cell
Hsp70 Cycle
• Co-chaperones: proteins that bind to Hsp70 and regulate its cycle
• Begin with opened Hsp70 in the ATP-bound form
o DNAJ proteins (co-chaperones) bind to Hsp70 transiently and active ATP
hydrolysis by Hsp70
• Hsp70 now in ADP-bound form, can bind substrate
• NEFs induce the exchange of nucleotide from ADP --> ATP
o Induce the release of ADP, so end up in no-nucleotide state
o In the cell, have excess of ATP compared to ADP, so ATP binds (increased
probability); back in opened state
• DNAJ stimulates ATP hydrolysis by Hsp70
• Hsp70-ADP binds substrate
• Nucleotide exchange factors (NEF) promote substrate dissociation
• DNAJs and NEFs increase the speed at which cycle takes place
o Relatively fast Hsp70 cycle when both present
• Some DNAJs can bind to substrate themselves, and when they activate ATP
hydrolysis by Hsp70, end up transferring the substrate from the DNAJ onto the
Hsp70
DNAJ (Hsp40) Co-Chaperones
• DNAJs regulate Hsp70 function
• Many DNAJs --at least 53 genes in human cells
o All have conserved J domain
• J domains: bind transiently to Hsp70, activate it to hydrolyze ATP and bind
polypeptide
o Do not bind substrate
• Induces substrate binding, like closing handle on plier
o Binds Hsp70, which causes a change that induces ATP hydrolysis
DNAJ Specificity
• J domain contacts Hsp70
• In addition to J domain that binds to Hsp70, DNAJs contain other domains that
determine its specific biological function
o Extra domains can be used to bind unfolded polypeptide substrates
• Some DNAJs bind substrate through specific domains, and act as ATP-
independent chaperones
o Substrate-binding activity themselves
o Do not use ATP, only use ATP through Hsp70
• Some DNAJs do not bind substrate
o Attach protein to complex of other proteins or organelle
• Recruit Hsp70 to that particular organelle to do specific function
o Specific DNAJ domains attach it to a protein complex or intracellular
membrane
o These DNAJs recruit Hsp70 to the complex or membrane
Substrate Binding DNAJ
• Substrate-binding DNAJs are the most highly conserved
• J domain, substrate binding domain, dimerization domain
• Homodimers: 2 subunits of 40-50 kDa --originally identified as Hsp40
o Dimerization means two subunits coming together
o Dimerization site at C-terminus
• Bind short hydrophobic sequences
o Bind sequences, recruit Hsp70 and then transfer that polypeptide to Hsp70 so
must bind the same type of sequence
• Transfers substrate to Hsp70 during ATP hydrolysis
Hsp70 NEFs
• Nucleotide Exchange Factors remove ADP from Hsp70 and allow ATP to bind
o ATP binds to nucleotide-free state
• Do this through physical event
o NEF binding physically opens up Hsp70 ATPase domain and weakens
interaction with nucleotide (ADP will fall out)
o ATPase domain of Hsp70 seen in orange
• ATP binds when NEF dissociates
o NEF leaves, allowing ATPase domain to close again and so ATP can bind
• ATP-bound Hsp70 releases polypeptide
• NEF binds, ATPase domain opens, Hsp70 releases ADP, NEF leaves, ATP binds
• Several NEF families in humans
o All do essentially the same thing; open up Hsp70 ATPase domain to allow
ADP to come out
How Does Hsp70 Help Folding?
• Hsp70 binds hydrophobic regions of folding intermediates and prevents incorrect
contacts from forming
o Prevents protein aggregation by covering up exposed hydrophobic regions
• On the other hand, these exposed hydrophobic bits have to end up inside the
protein in the native state
o So protein cannot fold until Hsp70 lets go
o Release of polypeptide from Hsp70 provides chances for it to fold
• Balance between DNAJs & NEFs supports an optimal rate of Hsp70 substrate
binding and release
o Gives fast cycles of binding & release
o Both steps of the cycle needed for Hsp70 to promote folding --need to bind to
prevent aggregation and to release such that protein can fold
• Substrate-binding DNAJs may provide additional assistance
o Second binding site such can bend and twist (need two points of contact)
o Both Hsp70 and DNAJs bind the polypeptide in transient state
• Can form multi-chaperone complex with Hsp90
o Hsp70 and Hsp90 can cooperate
Hsp90 Family
• Hsp90 chaperones are homodimers with 2 identical subunits joined at the C-
terminus
o Human Hsp90: 2 x 90kDa = 180kDa
o Very different from Hsp70 structurally (different tool)
• N-terminal domain binds ATP
o Middle/C-terminal domain form the dimer
• Dimer can open & close, like nutcracker
o ATP controls opening and closing of the dimer
• Opens and closes at the hinge on the C-terminus, part of functional cycle
Hsp90 Cycle
• ATP binding allows dimer to close
• Substrate is weakly bound in the open, nucleotide-free (apo) state
o Tightly bound in the closed ATP-bound state
• ATP hydrolysis to ADP compacts the dimer and releases the substrate
o Compact when lose phosphate and open when lose ADP
• Start in the open state, nucleotide weakly bound (apo)
o All ATPases have 3 states: ATP-bound, ADP-bound, and no nucleotide
• Binds ATP; ATPase domain changes its conformation
o Allows Hsp90 to close
o Begins to bind substrate in first state, but binds substrates most stably in the
closed ATP-bound form
• ATP hydrolysis induces other conformational change, in which subunits tightly
bound to each other
o Pushes the polypeptide substrate off Hsp90
• Release of ADP causes return to initial open state
• Binding substrate in ATP-bound state, unlike Hsp70
