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