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BMB170Lecture11NucleicAcids,Oct.31
• DNA– Transcriptional regulators:
repressors, activators– Enzymes to cut, modify or replicate
DNA– Histones
• RNA– Large complexes (Ribosome, SRP,
spliceosome)– tRNA synthetases– Translation factors
Types of proteins that bind to Nucleic Acids
‘StructuresoftheCRISPRgenomeintegraDoncomplex’Wrightetal(DoudnaLab)Science(2017)357:1113
Some classes of DNA binding transcription factors
• HTH (helix-turn-helix) proteins• Homeodomains• Steroid receptors• Zinc finger proteins• Leucine zipper proteins• Helix-loop-helix proteins• β-sheet motifs
• Each motif involves simple secondary structure that is complementary to B-DNA. Side chain contacts allow same motif to be used for recognizing different DNA sequences.
WhatsecondarystructuresofproteinsarecomplementarytoB-DNA?
• MostproteinsdecodesequenceinformaDonfromDNAmajorgroove.• α-helixfitsintomajorgrooveofB-DNA• Two-strandedanDparallelβ-sheetcanfitintominorgrooveofB-DNA(Church
etalPNAS(1977)74:1458-)
5.8Å
13.6Å
6.7Å
9.6Å
Helix-turn-helix motifs in DNA binding proteins
• Conserved recognition motif
• First seen λ cro, E. coli CAP, λ repressor.
• Sequence comparisons suggested HTH motif occurs in large family of prokaryotic DNA binding proteins (find highly conserved glycine and several hydrophobics).
• HTH occurs in different structural environments
Repressors control lytic vs lysogeny decision in phage
• Phage two parts lifecycle– (1) Prophage
• phage genome incorporated• want lytic genes off
– (2) Lytic • turn off repressor of lytic genes• turn on lytic genes
• Prophage state (as diagrammed)– cI (Clear 1 - λ repressor) binds to OR1 and OR2– turns off PR by blocking RNA pol from transcribing lytic genes (e.g., cro)– cI binding turns on PRM to make more cI (cI is both a repressor and an activator).
• Lytic state– DNA damage leads to cI cleavage– cro (control of repressor operator) binds to OR3, turns off PRM– turns on PR to transcribe lytic genes.
• cro and cI bind to same operators but with differing affinities:– cI: OR1 > OR2 > OR3– cro: OR3 > OR2 > OR1
~17 bp operators are nearly palindromic
Prophage state
cI
Structure of λ cro protein from phage lFirst structure of a DNA binding protein (Matthews lab, 1981) (5cro)
• 66 aa long• Binds as dimer to 17 bp
pseudo-symmetric operator
• Dimer made by β-sheets• HTH motif is 2nd and 3rd α-helices
• Model for λcro/DNA – two copies of the
recognition helix separated by 34 Å
– same distance as separates two major grooves of B-DNA.
~34Å
Anderson et al. Nature (1981) 290:754-8
α1α2 α3
Structure of DNA-binding domain of cI (λ repressor) (1lrp)
• cI has two domains– N-terminal 92 res DNA binding– C-terminal stabilizes dimer
• DNA-binding domain structure– HTH motif is helices 2 and 3. – Dimer contact is mediated by helix 5
• Recognition helices separated by ~34 Å (λ cro) • N-terminal arms reach around to back side of DNA to
make contacts with major groove.
Pabo and Lewis, early 80’s. C Pabo & M Lewis Nature (1982) 298:443-7
2
3
2
3
cI (λ repressor) bound to 20-mer oligo (1lmb)
• Protein dimer symmetry axis coincides with approximate two-fold axis of DNA oligo.
• Recognition helices on adjacent major grooves.
• N-terminal arms contact major grooves on back of DNA.
• DNA slightly distorted from B-DNA.
Beamer & Pabo JMB (1992) 227:177-96.
HTH
Structural conservation of HTH motif
• Cα’s superimpose within 0.7 to 1.0 Å rmsd
• 6/21 aa conserved in related sequences
• 4 residues make hydrophobic contacts between helices, preserving their orientation
• Conserved Gly important for bend between helices
1st helix
2nd helix(recognition helix)
Phage 434
• Similar to λ phage• Structures of
repressor and Cro– Fold overall similar– Similar DNA binding
modes• Bends DNA
434 Cro (3cro)
434 repressor (2or1)
Harrison lab:Aggarwal et al Science (1988) 242:899-907 Wolberger et al Nature (1988) 335:789-95
HTH HTH
CAP/cAMP/DNA structure(1cgp)
Steitz lab: McKay & Steitz Nature (1981) 290:744-9Schultz et al Science (1991) 253:1001-7
• cAMP receptor protein• Activates at over 20
promoters• w/o DNA predicted to
bind left-handed DNA • Bends DNA by 90°
CAP/cAMP structure
Comparison of l repressor and 434 repressor/DNA complexes
• 1st residue of 1st helix (Gln) – two H-bonds with DNA backbone– Aligns +helix dipole of 1st helix with phosphates.
• 1st residue of 2nd helix (Gln)– makes bidentate H-bonds to adenine – Gln is specific for A of A-T base pair
• H-bonds between Glns at beginning of each helix– Stabilizes geometry and dipole interactions– No code - simple mutagenesis schemes to change
specificity won’t work• Asn at end of recognition helix H-bonds to same
Pi oxygen contacted by first Gln
Harrison lab: Pabo et al Science (1990) 247: 1210-3
Comparison of λ repressor and 434 repressor/DNA complexes
Trp repressor
• TrpR/W (1tro) white/blue-red – turns off W synthesis– Trp moves HTH motif (D and E)
• Apo TrpR (3wrp) shaded/gray– HTH orientated incorrectly– apo TrpR can’t bind B-DNA Sigler lab: Zhang et al (1987) Nature 327: 591-597
TrpR/DNA complex (2.4 Å)
• Trp R/DNA (19-mer oligo)• Water mediated H-bonds• 24 direct, 6 solvent-mediated
H bonds to Pi backbone• Sequence recognized
indirectly through effects on geometry of Pi backbone
• No sequence specific vdw contacts between non-polar sidechains and bases
• Crystals grown in 35% dimethylpentanediol (1 year)
B DNATrp R-bound DNA
Sidechains that make directH-bonds to the operator
Sigler Lab: Otwinowski et al (1988) Nature 335: 321-9 (1tro)
Anon-specificcomplex?
Staacke et al (1990) How Trp repressor binds to its operator. EMBO Journal 9: 1963-7
• TrpR binds three operators (trpR operon, aroH operon, operon for Trp synthesis)
• Consensus binding sequence unusually large (protection experiments)
• Crystal structure of TrpR and oligo with consensus sequence of trp operators about a central axis of symmetry
• Propose that operator binds two TrpR dimers on full sequence
Two TrpR dimers (1trr)
• Crystal structure with the larger oligo
• Binds a dimer• Confirms Sigler model
of water mediated contacts to DNA
Lawson & Carey (1993) Nature 366:178-82
Phillips Lab: Rafferty et al (1989) Nature 341:705-710
Met Repressor (1cmc)
SAM
• Methionine is precursor to S-adenosylmethionine (SAM)• Binds SAM• Solved structure +/- SAM• Dimer of two highly intertwined monomers. No HTH motif. • No change in structure upon SAM binding
C
C
~35Å
Somers & Phillips (1992) Nature 359:387-93
Met repressor/DNA complex (1cma)
• A two-stranded β-sheet inserts in major groove
• Anti-parallel β-ribbon has two-fold axis and twist curvature is comparable to that of DNA
Homeodomains - eukaryotic HTH motifs
recognitionhelix
recognitionhelix
1st helixof HTH motif
1st helixof HTH motif
Branden and Tooze, Fig. 9.9
• First discovered in Drosophila proteins that regulate development• Bind “homeoboxes” • Large family of proteins that regulate transcription• Homeodomains are stably folded domains rather than motifs• Bind to AT-rich regions• Primary sequences highly conserved• Sequence comparisons suggested HTH motif -- verified by structures• N-terminus forms arm that inserts into minor groove
Q50
I47
N51
R3
R5
Engrailed/DNA structure (1hdd)
• engrailed involved in Drosophila development
• HTH motif: superimposes on prokaryotic repressors to 0.84 Å rmsd
• N-terminal arm (residues 3-9) fits in minor groove
• Three helices– 1&2 are anti-parallel, no DNA contacts– Helix 3 ~90°to first two, fits major groove Pabo lab: Kissinger et al (1990) Cell 63: 579-90
Combinatorial control of gene regulation• Homeodomain transcription factors
– low DNA binding specificity – Bind in combination with factors– increases binding affinity & specificity
• Combinatorial control–modular combination of a limited number of factors– control expression of a variety of genes
• Example: Mating type in yeast–MATα2 (a homeodomain-containing protein) binds to DNA
together with either a1 or MCM1– Diploid cells: MATα2 + a1 represses haploid-specific genes
Haploid cells : MATα2 + MCM1 represses diploid-specific genes
– Crystal structures of MATα2/DNA, MATα2/ a1/DNA, and MATα2/MCM1/DNA
Matα2/DNA structure (1apl)Pabo lab: Wolberger et al Cell (1991) 67:517-28
• Homeodomain of MATα2 bound to DNA– Similar to engrailed/
DNA complex– N-terminal arm
interactions with minor groove.
• Residues C-terminal to recognition helix are disordered.
• a1 is a homeodomain• C-terminal tail of MATα2 – disordered in MATα2/DNA – ordered (gray) in MATα2/
a1/DNA – Packs against a1 as an
amphipathic helix• DNA is bent
MATα2/a1/DNA structure
MATα2
a1
Wolberger lab: Li et al (1995) Science 270:262-9 (1YRN)
Matα2/MCM1 &MADS box
• Haploid cells, Matα2 interacts with MCM1 to repress a-specific genes
• MCM1: 286 residues – N-terminal 80 aa domain similar to
mammalian Serum Response Factor – specifies DNA binding, dimerization,
and interaction with accessory factors.
• Contains a SRF-like domain is 56 aa MADS-box– shared by mammalian myocyte
enhancer factor 2 (MEF2) transcription factors
– plant homeotic genes Agamous and Deficiens
Serum Response Factor (1srs)Pellegrini, Tan & Richmond (1995) Nature 376:490-8
Matα2/MCM1/DNA structure
• Binding of MATα2 to DNA– MCM1 increases affinity 50-500 fold– spacing must be correct.
• Cooperative binding – interaction between MCM1 and flexible linker region of
MATα2.• Crystal structure 2.25 Å
– homeodomain from MATα2– MADS-box transcription factor MCM1– DNA
Tan & Richmond Nature (1998) 391: 660-6
α2 (cis)
α2 (trans)
MCM1 (cis)
MCM1 (trans)
STE6 DNA
Matα2/MCM1/DNA structure (1mnm)
•MCM1 is dimeric (similar to SRF/DNA crystal structure)•Long helices of MCM1 nearly parallel to minor groove -- they extend into major groove.•N-terminal arm of MCM1 passes over DNA backbone.•N-terminal arm of MATα2 contacts minor groove, but more residues are ordered.•MATα2(cis) - ordered residues make strands S1 and S2, helps bend DNA
Tan & Richmond Nature (1998) 391: 660-6
Steroidandthyroidhormonesuperfamily• Steroidandthyroidhormonesacttocoordinatecomplexeventsindevelopment– e.g.removethyroidfromtadpole
• LigandresponsivetranscripDonfactors– Hormonesbindtoreceptorsinsidecell– ReceptorsthenenternucleustoacDvategenes
• ReceptorsbindtoHormoneResponsiveElements(HREs)– 20bpcis-acDngsequencesrequiredforhormonalregulaDon– CanputHREinfrontofothergenestoregulateinresponsetohormone
• HREsareposiDonandorientaDonindependent• HREsaredyadsymmetric-dimers
• A protein that interacts with hormones– e.g. sex hormones, glucocorticoids,
thyroid hormone– binds to an enhancer to stimulate
transcription• Three typess
– Type I - glucocorticoid• reside in cytoplasm• migrates to nucleus when bound to
hormone– Type II - thyroid hormone
• reside in nucleus• binding in the absence of hormone
can repress transcription; binding with hormone stimulates transcription
– Type III - orphan receptors • ligand has not been identified
Nuclear Receptors
Steroid/thyroid receptors have separate DNA-binding and hormone-binding domains
Results of many biochemical studies showed these receptors have three domains: one involved in activation,one for DNA binding, one for hormone binding.
We’ll look at structureof DNA-binding regiononly.
• DNA binding domains have basic residues and conserved Cys
• Proposed that Cys residues coordinate zinc. Find two zinc- binding modules in each DNA-binding region. All DNA- binding regions have two zinc-binding modules.
• Position of Cys residues similar to TFIIA motifs, but no histidines (see next lecture)
DNA binding domains of steroid/thyroid receptors
Crystal structure of glucocorticoid receptor/DNA complex
• Crystallized with oligo with two hexameric half sites.• Half sites normally separated by 3 BP (pseudo two-
fold symmetry), but their oligo has 4 BP separation (true two-fold symmetry).
• One specific, one non-specific complex.Sigler lab: Luisi et al (1991) Nature 352: 497-505
• GR DNA-binding domains – bind as dimer– each chain has two Zn modules.
• Zn modules fold together as part of a larger globular domain
• Modules are not independent structural units
• Dimerization interactions force one subunit to interact with non-cognate sequence.
Glucocorticoid receptor/DNA complex (1r4r)
Sigler lab: Luisi et al (1991) Nature 352: 497-505
Glucocorticoid receptor/DNA complex (1r4r)
Sigler lab: Luisi et al (1991) Nature 352: 497-505
The two zinc-containing modules differstructurally and functionally.
Module 1: Module 2: Contains recognition helix. Contacts phosphates, dimerizes with partner.
Requires zinc for folding and binding DNA, but verydifferent from TFIIIA-type zinc fingers.
TFIIIA-style zinc fingers• Transcription factor IIIA (TFIIIA)
– Prototype Zinc finger protein– from Xenopus oocytes– required for accurate transcription of 5S RNA genes by RNA pol III– Purified protein binds Zn– Zn necessary for specific DNA binding
• cDNA sequence had 9 tandem sequences:Y F X C X2-4 C X3 F X5 L X2 H X3-4 H X2-6 – Called these sequences “fingers”– Each finger is structurally independent domain (protease digestions)– Each finger encoded on a separate exon
• 30 aa synthetic peptides bind zinc• Peptides are unfolded unless zinc added (CD experiments)
Some TFIIIA-style zinc finger proteins• Yeast ADR1 (alcohol dehydrogenase regulation) -- 2 fingers• Human SP1 (general transcription factor regulating cellular
and viral genes)• Krüppel, Hunchback (both involved in control of Drosophila
development• ZFY, ZFX (testis determining factor, found on X and Y
chromosomes)• Xenopus Xfin (37 fingers!)• 897 human proteins have at least one C2H2 Zn finger for a
total of 6890 C2H2 domains (~8 fingers/protein)(Ali Mortazavi, 2004 BMB 170 project)
Model of zinc finger (J. Berg)
Conserved residues circled
Model of zinc finger proposed by Jeremy Berg
J. Berg PNAS (1988) 85:99-102
• Berg Model– Binds Zn through invariant Cys and
His residues (EXAFS)– Two Cys hypothesized to interact
with Zn in same way as seen in rubredoxin and ATCase (a β-hairpin)
– Two His hypothesized to interact with zinc like His residues in hemerythrin and thermolysin (α-helix)
• Structure confirmed overall Berg model• “This may constitute the first time that a
globular protein structure has been correctly predicted from its amino acid sequence (depending on what one means by ‘correct’, ‘predict’, and ‘first’).” Tom Steitz Q Rev Biophys (1990) 23:205-80
Zinc fingers bind as modules to adjacent sites on DNA
Wright lab: Lee et al Science (1989) 245:635-7 (1znf)
• Each finger interacts with 3 bp (e.g., Sp1 site is 9-10 bp for binding three fingers)
• Methylation interference suggested major groove binding
• NMR structure– 31st Xfin finger– Exposed face of helix showed
basic residues and polar side chains
First x-ray structure: Three fingers from Zif268 (1zaa)
• Each finger contacts 3 bp in major groove.• Fingers are similar (0.45 Å to 0.87 Å rmsd).• α-helix
– N-terminus points into major groove– helix axis not aligned with major groove
• β-strands– 1st - no contacts with DNA– 2nd - contacts phosphate backbone
Pavletich & Pabo Science (1991) 252: 809-816
First X-ray structure: Three fingers from Zif268 (1zaa)
• Arg reads the guanines• Majority of interactions to one strand (not
always the case)
Pavletich & Pabo Science (1991) 252: 809-816.
5-finger complex from hGLI oncogene (1gli)
Pavletich & Pabo Science (1993) 261: 1701-7
Leucine zippers
• First discovered in 30 aa segment of C/EBP (enhancer binding protein)
• Found Leu every 7 residues over 8 helical turns• 1st model (Landschultsz et al Science (1988)
240:1759-64) – Proposed both parallel and anti-parallel– Favored antiparallel coiled coil
• align helical dipoles favorably• allows leucines to interlock to form a “zipper”.
bZIP - Leucine zippers
• Leu region is parallel coiled coil– mutational & x-linking studies– Dimerization motif
• Basic region interacts with DNA• Can form hetero and homo dimers
– allows combinatorial action of gene regulatory proteins– doubles DNA contact area (squares affinity constant)
• Leucine zipper not really a “zipper”, but a parallel coiled coil– helices distorted (repeat is 3.5 residues/turn instead of 3.6)– integral repeat of Leu every 7 residues along helix
• Scissors grip model for bZIP proteins– zipper must be contiguous with basic region– basic region contacts DNA at bifurcation point of two zippered
helices
Structure of GCN4, a bZIP protein (1YSA)
• 56 aa bZIP element of GCN4 bound to 20 bp oligo
• single continuous α-helix of 52 residues
• C-terminal ends form coiled-coil• dimerization region ~perpendicular
to DNA• N-terminal basic-region splays
apart
Harrison lab, Ellenberger et al., 1992, Cell 71: 1223-1237
Helix-loop-helix proteins, another dimerization motif
• HLH portion responsible for dimerization• N-terminal basic region binds DNA• Family includes some proteins with no basic region
– no DNA binding– act as negative regulators of HLH proteins with basic regions.
• Myc oncoproteins have basic and HLH motifs followed by Leucine zippers (b/HLH/Z)
MyoD bHLH Domain (1mdy)
• Transcriptional activators in muscle cells• Binds to consensus CANNTG• Structure of dimer is a parallel, four helix bundle
Pabo lab, Ma et al (1994) Cell 77:451
Basic region
Basic region
Structure of Max, a b/HLH/Z protein, bound to DNA (1an2)
• Max is a b/HLH/Z protein that heterodimerizes with Myc oncoproteins• Association with Myc in vivo is required for malignant transformation• First bHLH fold
22 bp oligoN
C
Burley lab, Ferre-D’Amare et al (1993) Nature 363:38-45
Leu Zipper
HLH
basic
Nuclear factor kappa-B (NFκB)• First identified as DNA binding protein that
binds to site in Ig κ light chain enhancer.• Prototype of family of transcription factors
that have 300 aa Rel homology region (RHR)
• Members of RHR family– Homo or heterodimers– bind to κB sites in enhancer regions of
genes involved in cellular defense mechanisms and differentiation.
• RHR is at N-terminus responsible for dimerization, DNA binding, nuclear localization
• Highly variable C-terminal domains are responsible for transactivation
Crystal structure of RHR from NFκB p50 homodimer bound to idealized palindromic κB target
• “Butterfly” with protein domains as wings attached to cylindrical body of DNA• Contacts with DNA formed by loops between β-strands• No helical or sheet structure at recognition surface
Sigler lab (1nfk) Ghosh et al Nature (1995) 373: 303-10; Harrison lab (1svc) Müller et al, ibid, 311-7
Note protein wraps around DNA to make contacts all along major groove
IκBα/NFκB inhibited complex
• IκBα contains ankyrin repeats
• IκBα binds to the NLS preventing transport
• Blocks DNA binding• p50/p65
transcription factor
p65
IκBα
p50
Harrison lab, Jacobs & Harrison (1998) Cell 95:749-58 (1nfi)
NFAT/Fos-Jun/DNA co-crystal structure
• NFAT proteins are cytoplasmic in resting T cells• TCR is stimulated
– increases [Ca2+]– calcineurin (a phosphatase) dephosphorylates sites on NFAT
• Dephosphorylated NFAT imported into nucleus • Full response at NFAT sites require
– activation of members of AP-1 transcription factor family– AP-1 site just downstream of NFAT site in the promoters of IL-2 and
other cytokines (T-cell proliferation)• The immunosuppressive drugs cyclosporin A and FK506
are calcineurin inhibitors preventing import of NFAT into nucleus
Harrison lab: Chen et al (1998) Nature 392: 42-8
NFAT/Fos-Jun/DNA
• AP-1 heterodimer - Fos and Jun (both bZIP proteins)• NFAT (nuclear factor of activated T cells) has RHR DNA-binding region• Structure bZIP parts of AP-1 and RHR of NFAT bound to DNA fragment
from Interleukin-2 promoter• NFAT binds as a monomer, other RHR members (e.g., NFκB) are dimers
Harrison lab (1a02) Chen et al (1998) Nature 392: 42-8.
FosJun
NFAT