Download Mechanisms of Unidirectional Translocation & Unwinding

Helicases
as Molecular Motors
BIOC/MMG 352
Scott Morrical
Dept. of Biochemistry
<[email protected]>
References
Levin MK and Patel, SS (2003) “Helicases as Molecular
Motors”, Chapter 7 in Molecular Motors (M. Schliwa, ed.),
WILEY-VCH Verlag GmbH & Co, KgaA, Wenheim, pp. 179203.
Eoff RL and Raney KD (2005) “Helicase-catalysed translocation
and strand separation”, Biochemical Society Transactions 33,
1474-1478.
Korolev S, Yao N, Lohman TM, Weber PC, Waksman G (1998)
“Comparisons between the structures of HCV and Rep helicases
reveal structural similarities between SF1 and SF2 superfamilies of helicases. Protein Sci. 7, 605-610.
Lohman TM, Thorn K, and Vale RD (1998) “Staying on track:
common features of DNA helicases and microtubule motors”,
Cell 93, 9-12.
Helicases are…
…linear motor proteins that couple energy
from NTP hydrolysis to translocate along a
polynucleotide lattice.
DNA helicases
RNA helicases
RNA/DNA helicases
“Screwdrivers of the cell”
DNA Helicases
Translocate on DNA lattices.
Unwind duplex DNA to form ssDNA
intermediates required for DNA replication,
recombination, repair.
Process, translocate branched DNA structures-Holliday junctions, D-loops, etc.
DNA Translocases: Translocate w/o unwinding.
“DNA pumps”-- conjugation, viral packaging.
Turnover of protein-DNA complexes.
RNA Helicases
Translocate on RNA lattices.
Destabilize RNA secondary structure; promote
ribosome assembly, translation, RNA splicing,
editing, transport, & degradation.
RNA/DNA Helicases
Unwind RNA/DNA hybrids; transcription
termination, regulate DNA replication initiation,
etc.
Helicases & Human Disease
Also… Many viruses encode their own specific helicases.
DNA Helicases in the Replisome
DNA Replication Fork
DNA Flow in the E. coli Replisome
Replisome in Motion (zoom out)
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Replisome in Motion (zoom in)
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DNA Helicases in Recombination
Stalled DNA
replication forks
Holliday
junction
equals
RuvAB*
*E. coli RuvAB Holliday junction translocase
Translocation of Holliday Junctions by RuvAB
Helicase in Homologous Recombination & DNA Repair
RuvB
hexamer
RuvB
hexamer
RuvA tetramer
Basic Properties of Helicases
Nucleic acid-stimulated NTPase (Mg++ dependent)
Unidirectional translocation and unwinding:
5’ --> 3’
or
3’ --> 5’
Oligomeric structure (or lack thereof):
some are monomeric molecular motors
dimers, hexamers common
other uncommon forms observed
Helicase Polarity
Most helicases require a stretch of ssNA of specific polarity
adjacent to the duplex region to initiate strand separation.
5’ --> 3’ helicase:
5’
NTP
NDP
5’
Helicase Polarity (cont’d)
Some helicases require both 5’ and 3’ non-complementary tails
to initiate unwinding:
5’
NTP
3’
NDP
5’
3’
(This is common among hexameric ring helicases found at
replication forks.)
Rarely helicases can unwind from a blunt end:
5’
NTP
NDP
3’
i.e. E. coli RecBCD enzyme which has 2 helicase subunits (one
3’ --> 5’ and one 5’ --> 3’) that can simultaneously engage both
5’ and 3’ ended strands from a blunt dsDNA end.
RecBCD
Helicase Superfamilies
Amino acid sequence homology reveals conserved helicase motifs
including Walker A and B (motifs I and II, respectively).
Variations allow grouping into several related families and
superfamilies:
SF1
SF2-- DEAD, DEAH, DexH families named for
variations in Walker B
RecQ
SF3
Hexameric ring-- 6 identical (most) or non-identical (MCM
helicases) subunits
Family members probably share a general mechanism of
translocation and unwinding; however there is no correlation
between sequence and polarity or substrate specificity.
Helicase Structure
• All helicases share a RecA fold.
• Each helicase molecule contains a single NTP
binding site and a distinct polynucleotide binding
site.
These sites are allosterically linked, since
the NTPase activity modulates nucleic acid
binding affinity, and vice-versa.
Structure of Hepatitus C Virus (HCV) Helicase (SF2)
1A
2A
3
1A
2A
3
Rep (SF1) vs. HCV (SF2) Helicases
Conservation of structure and relationships of conserved helicase motifs
despite little overall sequence homology.
Rep
HCV
Sequence Alignments of the SF1 and SF2 Superfamilies
Based on Structural Super-Imposition
Common Features of SF1 and SF2 Helicase Structure
• Sub-domains 1A and 2A contain RecA-like folds.
• Residues from conserved helicase motifs line the interface
of 1A and 2A and bind NTP.
• ssNA binds in a groove that is formed by 1A and 2A subdomains.
• Motifs I, II, and III form NTP binding site while motifs V
and VI are positioned so as to help transmit a
conformational change from the NTP binding site to the NA
binding site.
• Domain movements between 1A and 2A as well as rigid body
rotations of other sub-domains have been implicated as
intermediates in helicase mechanisms.
Structure of
Helicase Domain of
Bacteriophage T7
gp4 Helicase
T7 gp4 Helicase
Structure (cont’d)
(C) Stereo view from inside the
ring of T7 gp4 helicase subunits A
and B complexes to ADPNP (dark
blue) and Mg++ (green, space
filling). The helicase conserved
motifs are shown in the same
color as in (B). The subunits are
colored the same as in (A). Amino
acid residue R522 is shown.
Common Features of Ring Hexamer Helicase Structures
• Helicase domain fold is RecA-like.
• Conserved helicase motifs and NTP binding site are at
subunit interface.
• A critical arginine residue (R522 in T7 gp4 helicase) from a
neighboring subunit is within hydrogen-bonding distance of the
gamma phosphate of NTP bound at the interface, and is
implicated in transducing conformational changes between
subunits of the hexamer.
• The central channel of the ring is large enough to
accommodate a single strand of DNA or RNA.
• Rings appear to adopt multiple asymmetric conformations in
response to ligand binding.
Mechanisms of Unidirectional Translocation & Unwinding
• Unwinding is a combination of unidirectional translocation and
strand separation processes.
• Both processes require energy from NTP hydrolysis.
• NTP binding, hydrolysis, and product release act as a switch that
induces conformational changes on the helicase NA binding site.
Conformational changes force the helicase to alter its
NA-binding affinity or to perform a power stroke.
Conformational changes drive unidirectional translocation
and unwinding in a step-wise fashion.
Mechanisms of Unidirectional Translocation & Unwinding:
Stepping Models
Stepping requires at least 2 NA binding sites that independently bind and
release NA and change the distance between each other.
• Monomeric helicases: 2 hands represent 2
parts of NA binding site that move relative to
each other. Both sites are controlled by a
single NTPase site.
• Oligomeric helicases: hands represent NA
binding sites on 2 different subunits.
Coordinated NTPase activity at multiple sites
leads to coordinated binding/release of NA.
Mechanisms of Unidirectional Translocation & Unwinding:
Stepping Models (cont’d)
• Helicase undergoes a round of conformational changes (i to vi) after which it appears
in the same conformational state (i’) one step away from its original position.
• Cycle starts with 1st NA binding site tightly
bound to NA (closed hand, i) and 2nd site
weakly bound to NA (open hand).
• In a power stroke motion, the 2nd site moves
away from the 1st (ii), followed by tight binding
of the 2nd site (closing, iii), opening of the 1st
site (iv), a power stroke bringing sites back
together (v), closing of the 1st site (vi), and
finally opening of the 2nd site (i’).
A Stepping Model Proposed for Dimeric Rep Helicase
Note that Rep actively engages duplex to promote unwinding.
Mechanisms of Unidirectional Translocation & Unwinding:
Brownian Motor
Use thermal fluctuations from surroundings to achieve unidirectional movement,
biased by NTP hydrolysis.
• Helicase has a periodic dependence of binding energy along the
length of ssNA (saw-tooth)
• If saw-tooth is asymmetric as
shown, then most helicase-ssNA
binding events (i) will cause leftto-right movement, bringing
helicase to its local minimum (ii)
where it is trapped.
Mechanisms of Unidirectional Translocation & Unwinding:
Brownian Motor (cont’d)
Use thermal fluctuations from surroundings to achieve unidirectional movement,
biased by NTP hydrolysis.
• Now suppose that NTP binds
and lowers helicase-ssNA binding
energy and makes it uniform
along the length of the ssNA
(dotted line).
• This allows the helicase to
slide along ssNA randomly in
either direction because of
Brownian motion (iii & iv).
• After NTP hydrolysis and product release, the asymmetric saw-tooth profile returns and
helicase binds to ssNA tightly. If Brownian motion moved the helicase backward (iii) it will
end up in the same position it started in and there is no net movement (ii).
• However if Brownian motion moves the helicase forward (iv) it will end up one step
forward after NTP hydrolysis and product release (ii’).
Factors Influencing Brownian Motor Efficiency
Efficiency = fraction of productive steps / NTP hydrolyzed
• The greater the assymetry of the sawtooth, the greater the
efficiency, since it will increase the fraction of helicase
molecules that reach poisiton iv and make a step forward.
• Efficiency depends strongly on rate of ATP hydrolysis, which
determines the lifetime of the weakly bound state (iii & iv).
• A helicase can move forward even against a moderate external
force as long as a significant fraction of the helicase can diffuse
forward to position iv.
Stepping vs. Brownian Motors
• Stepping requires at least 2 independent NA binding sites that move relative
to each other.
• A Brownian motor with only 1 NA binding site can still move unidirectionally.
• Stepping-- force is generated at the junction of the NA binding sites. This
force moves the two binding sites relative to each other, generating
unidirectional movement.
• Brownian-- 2 kinds of forces: One is generated at the NA binding site during
tight binding of helicase to NA. The other is Brownian motion that can move the
helicase in any direction along the NA while the helicase binding is in a flat
energy profile.
• Brownian model predicts limited processivity for monomeric helicases.
• Dimeric or hexameric helicases with 2 or 6 ssNA binding sites can implement a
Brownian mechanism more efficiently, especially if NTPase cycles are
coordinated between subunits.
Mechanisms of Unidirectional Translocation & Unwinding
Estimating Step Size and Processivity
• The step size of a helicase can be estimated from single
turnover kinetics of unwinding reactions and fitting to a stepping
equation.
• The lower limit step size can be determined by measuring the
coupling ratio (number of bases traveled per NTP hydrolyzed).
• Processivity (P) = probability of making a step forward on a NA
lattice divided by the probability of dissociation from that position on
the lattice.
Monomeric helicases:
UvrD, PcrA
P ≈ 0.9 (10 bp)
Hexameric helicases:
T7 gp4
P ≈ 0.99996 (33 kbp)
Mechanisms of Unidirectional Translocation & Unwinding
Strand Separation
Basepair hydrogen bond free
energies:
DG ≈ 10 kJ mol-1 base-1 at 15ºC
(unwinding thermodynamically
unfavorable)
Spontaneous opening rates:
1000 s-1 near ends
30 s-1 in middle
Mechanisms of Unidirectional Translocation & Unwinding
Strand Separation via “Active” vs. “Passive” Mechanisms
• To make unwinding thermodynamically favorable, helicases must stabilize
open bps by binding to ssNA one step at a time.
• Active mechanisms: helicase increases the rate of bp opening in addition to
stabilizing the ssNA product.
Rate can be increased by lowering transition state energy.
Specific binding to unwinding intermediate, i.e. distorted duplex region.
May be useful
for helicases
with large
step size.
• Passive mechanisms: by definition can’t change rate of bp opening.
Mechanisms of Unidirectional Translocation & Unwinding
Another View: Helicase as Translocating Wedge
• A helicase could in principle unwind dsNA by translocating between 2
strands of NA while pushing them apart without any specific interactions.
• Any protein capable of translocating along one strand of dsNA while
excluding the other can unwind dsNA by a wedge mechanism (see RuvAB).
T7 gp4 helicase shows little interaction with duplex, unlike
Rep or PcrA.
A classic wedge?
Note small step size (est 2 bp).
Replisome in Motion (zoom in)
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