HLH Motif and Leucine Zipper , domains - molecular biology

Another DNA binding domain, the Helix-loop-helix (HLH) dimer, is shown bound to DNA fragment — each alpha helix represents a monomer. Leucine zipper is created by the dimerization of two specific alpha helix monomers bound to DNA. The leucine zipper is formed by amphipathic interaction between two ZIP domains.

1. HLH Motif andLeucine
Zipper
SOUSAN
A274153121006
HG&MM

2. CONTENT
➢Introduction
➢HLHMotif
➢Leucine Zipper
➢Interaction
➢Conclusion

3. INTRODUCTION
➢Regulatory proteins have domains for DNA binding and protein-
protein interactions.
➢Examples include eukaryotic transcription factors functioning as gene
activators.
➢DNA-binding domains often contain zinc fingers.
➢Dimer formation is typically necessary for DNA binding.
➢Structural motifs facilitating protein-protein interactions include
leucine zipper and basic helix-loop-helix.
➢These motifs classify regulatory proteins into structural families.

4. HLH Motif

5. Helix-Loop-Helix
➢Eukaryoticregulatoryproteinsinmulticellularorganismdevelopment
haveacommonstructuralmotif.
➢Thismotifconsistsofaconservedregionofabout50aminoacid
residues.
➢ItiscrucialforbothDNAbindingandproteindimerization.
➢Themotifformstwoshortamphipathicαheliceslinkedbyavariable-
lengthloop.
➢Thishelix-loop-helixmotifdiffersfromthehelix-turn-helixmotif
associatedwithDNAbinding.
➢Dimerformationoccursthroughtheinteractionofthehelix-loop-helix
motifsoftwopolypeptides.
➢DNAbindingisfacilitatedbyanadjacentshortaminoacidsequencerich
inbasicresidues,similartoproteinscontainingleucinezippers.

6. STRUCTURE
➢The helix–loop–helix motif consists of a short
α helix connected by a loop (red) to a second,
longer α helix.
➢The flexibility of the loop allows one helix to
fold back and park against the other, forming
the dimerization surface.
➢Two-helix structure binds both to DNA and
to the two-helix structure of a second protein
to create either a homodimer or a
heterodimer.
➢Two α helices that extend from the
dimerization interface make specific contacts
with the major groove of DNA.

7. EXAMPLE
➢The human transcription factor Max, bound to its DNA target site.
➢The protein is dimeric; one subunit is colored.
➢The DNA-binding segment (pink) merges with the first helix of the
helix-loop-helix (red).
➢The second helix merges with the carboxyl-terminal end of the
subunit (purple).
➢Interaction of the carboxyl-terminal helices of the two subunits
describes a coiled-coil.
➢Similar to leucine zipper but with only one pair of interacting Leu
residues (red side chains near the top)
➢The overall structure is sometimes called a helix-loop-helix/leucine
zipper motif.

8. Inhibitory regulation by truncated
HLH proteins
➢The HLH motif is responsible for both dimerization and
DNA binding.
➢HLH homodimer recognizes a symmetric DNA sequence.
➢The binding of a full-length HLH protein (blue) to a
truncated HLH protein (green) that lacks the DNA-
binding α helix generates a heterodimer that cannot bind
DNA tightly.
➢If present in excess, the truncated protein molecule blocks
the homodimerization of the full-length HLH protein,
thereby preventing it from binding to DNA.

9. FUNCTION
➢bHLHtranscription factors are often important in
development or cell activity.
➢BMAL1-Clock is a core transcription complex in the
molecular circadian clock.
➢Other genes, like c-Mycand HIF-1, have been linked to
cancer due to their effects on cell growth and metabolism.

10. Basic Helix-Loop-Helix (bHLH)
➢Astructural domain of 50–60amino acids.
➢Forms two amphipathic α helices separated by an intervening loop.
➢Two separate polypeptides can interact to form a dimer that
bindsDNAas a parallel, left-handed, four-helix bundle.
➢Sequence-specific DNA recognition is mediated primarily by a stretch of
basic amino acids that reside near the amino-terminal flank of each
dimerized bHLHmotif
➢bHLHproteins typically bind to a consensus sequence called an E-box
CANNTG.
➢Since many bHLHtranscription factors are heterodimeric, their activity
is often highly regulated by the dimerization of the subunits.
➢One subunit's expression or availability is often controlled, whereas the
other is constitutively expressed.

11. Differences between HLH &HTH
Helix-Loop-Helix
➢Aprotein structural motif that
defines dimerizing transcription
factors.
➢Mediatesproteindimerization.
➢Group A, B, C, D, E, & F with
relevant transcription factors.
➢Containstwohelices, anda loop
connects them.
➢Sex determination and nervous
system and muscular
development.
Helix-Turn-Helix
➢A protein structural motif that is
capable of binding DNA.
➢MediatesDNAbinding.
➢Di, Tri, Tetra helical, and winged
helical.
➢Eachmonomer organizerswith
twoalpha helices joined by a short
amino acid strand binding to a
groove in DNA.
➢Regulationofgeneexpression.

12. Leucine Zipper

13. STRUCTURE
➢The motif is an amphipathic α helix with hydrophobic amino acid
residues concentrated on one side.
➢Hydrophobic surface forms the contact area between polypeptides in a
dimer.
➢Leu residues occur at every seventh position, forming a straight line
along the hydrophobic surface.
➢Initially thought to interdigitate, now known to line up side by side as
helices coil around each other, forming a coiled-coil.
➢Regulatory proteins with leucine zippers often have a separate DNA-
binding domain rich in basic residues.
➢Leucine zippers are found in many eukaryotic and a few prokaryotic
proteins.

15. LECINE ZIPPER
The leucine zipper motif is named because of how the
two α helices, one from each monomer, are joined to
form a short coiled coil. These proteins bind DNA as
dimers where the two long α helices are held together
by interactions between hydrophobic amino acid side
chains (often on leucines) that extend from one side of
each helix. Just beyond the dimerization interface, the
two α helices separate from each other to form a Y-
shaped structure, which allows their side chains to
contact the major groove of DNA. The dimer thus grips
the double helix like a clothespin on a clothesline

16. A leucine zipper dimer bound to DNA
➢Two α-helical DNA-binding domains (bottom)
dimerize through their α-helical leucine zipper
region (top) to form an inverted Y-shaped
structure.
➢A single α helix forms each arm of the Y, one
from each monomer, that mediates binding to a
specific DNA sequence in the major groove of
DNA.
➢Each α helix binds to one-half of a symmetric
DNA structure.
➢The structure shown is of the yeast Gcn4
protein, which regulates transcription in
response to the availability of amino acids in
the environment.

17. Heterodimerization Expands the Repertoire of DNA
Sequences Recognized by Gene Regulatory Proteins
➢Many gene regulatory proteins, including leucine zipper
proteins, can form heterodimers composed of two
different subunits.
➢Heterodimers are made from proteins with distinct
DNA-binding specificities.
➢This mixing and matching greatly expand the
repertoire of DNA-binding specificities.
➢For example, two types of leucine zipper monomers
could generate three distinct DNA-binding specificities.
➢With three types of monomers, six distinct DNA-
binding specificities could be created, and so on.

18. ➢Promiscuous heterodimer formation among leucine
zipper proteins could lead to excessive cross-talk in gene
regulatory circuits.
➢The extent of cross-talk depends on how well the
hydrophobic surfaces of the α helices of leucine zippers
mesh.
➢Each leucine zipper protein can only form dimers with a
small set of other leucine zipper proteins due to specific
amino acid sequences.
➢Heterodimerization exemplifies combinatorial control,
where combinations of proteins control cellular processes
rather than individual proteins.
➢It is one mechanism used by eukaryotic cells to regulate
gene expression.
➢Heterodimerization occurs across various types of gene-
regulatory proteins.
➢It is part of various combinatorial mechanisms for
controlling gene expression.

19. ➢Duringtheevolutionofgeneregulatoryproteins,similarcombinatorial
principleshaveproducednewDNA-bindingspecificitiesbyjoiningtwo
distinctDNA-bindingdomainsintoasinglepolypeptidechain.

20. FUNCTION
➢Mediating Dimerization
➢DNA Binding
➢Regulation of Gene Expression
➢Signal Transduction
➢Protein-Protein Interactions

21. CONCLUSION
➢In terms of the way it interacts with DNA, the helix–loop–helix motif is more closely related
to the leucine zipper motif than it is to the helix–turn–helix motif.
➢The helix-loop-helix (HLH) motif and the leucine zipper motif are both common in proteins
involved in DNA binding and regulation. While the helix-turn-helix (HTH) motif is another
DNA-binding motif, it's structurally different from both HLH and leucine zipper motifs.
➢The HLH motif typically consists of two alpha helices connected by a loop. It often forms
dimers, where two HLH-containing proteins come together, each contributing one helix to
form a functional DNA-binding unit. These proteins are often involved in transcriptional
regulation, where they bind to specific DNA sequences to either activate or repress gene
expression.
➢On the other hand, the leucine zipper motif involves a coiled-coil structure formed by two
alpha helices, where leucine residues at specific intervals on one helix interact with leucine
residues on the other helix. This motif also facilitates dimerization, and the dimeric
complex often binds to DNA.
➢Although they have distinct structural features, both HLH and leucine zipper motifs
facilitate protein dimerization, which is crucial for their DNA-binding function. This
dimerization enables the formation of stable complexes that can efficiently bind to DNA
sequences and regulate gene expression.

22. Question 1:
➢In the leucine zipper DNA binding domain the hydrophobicity
caused by
A)Presence of hydrophobic leucine residue at 7th position an N-
terminal
B) Presence of hydrophobic leucine residue at 7th position on C-
terminal
C)Presence of hydrophobic leucine residue at 7th position in turn
D)None of the following are the correct reason

23. Question 2:
➢Which one of the following combinations must be present in a steroid
receptor that is located in the cytoplasm?
(a)Nuclear Export Sequence (NES), leucine zipper
(b) NES, zinc finger motif
(c) NLS, leucine zipper
(d) Nuclear localization sequence (NLS), zinc finger motif

24. Question 3:
➢Phosphorylation of ADP to ATP occurs through energy metabolism,
comprising oxidative phosphorylation or substrate-level phosphorylation
or photo-phosphorylation (in plants). ATP can also be formed from ADP
through the action of adenylate kinase. Crystal structure determination
of adenylate kinase shows that the C-terminal region has the sequence -
val-asp-asp-val-phe-ser-gln-val-cys-thr-his-leu-asp-thr-leu-lys.
What can be a possible conformation of the sequence?
(1) A helix that is not amphipathic
(2) Amphipathic helix
(3) Leucine zipper helix
(4) Beta helix

25. THANK YOU
References:
▪Lehninger: Principles of Biochemistry, Chapter: Principles of Gene
Regulation
▪Molecular Biology by Bruce Alberts