3222
Biochemistry 1995,34, 3222-3230
Designing Zinc-Finger ADRl Mutants with Altered Specificity of DNA Binding to T in UASl Sequences? Wayne E. Taylor,*%'Hubert K. Suruki,§ Amy Hsiu Ti Lin," Pejman Naraghi-Arani,l Robert Y. Igarashi, Maziar Younessian,# Pamela Katkus, and Nam V. VoA Department of Chemistry and Biochemistry, California State University, Fullerton, California 92634 Received August 11, 1994; Revised Manuscript Received November 21, 1994@
Yeast ADRl contains two Cys2,His2 zinc fingers needed for DNA binding to the upstream activation sequence UAS 1, with bases T ~ T ~ G ~ - G ~ in A the ~ GADH2 Io promoter. Potential DNA-contacting amino acid residues at -1, +3, and +6 in the a-helical domains of ADRl's fingers one and two include RHR-RLR; however, the latter finger two residues Leu146 and Arg149 had not proved to be crucial for ADRl binding, even though Leu146-7'6 and Arg149-Ts interactions .with UAS 1 DNA were predicted. We altered Leu146 or Arg149 by PCR cassette mutagenesis, to study ADRl mutant binding to 16 UASl variants of thymine bases T5 and T6. Mutation of Leu146 to His, making finger two (RLR) like finger one (RHR), decreased binding to wild type UASl having T6, but enhanced its binding strength to sequences having purines G6 or A6, similar to binding seen between finger one's His118 and base As of UAS1. Mutating Leu146 to Lys caused this finger two RKR mutant to bind strongly to both G6 and T6, possibly by lysine's amine H-bonding to the carbonyl of guanine or thymine. Specificity of ADRl for UASl with T6 may thus be due to hydrophobic interaction between Leu146 and the T6 methyl group. ADRl mutants with either His or Lys in the central +3 residue (146) of zinc finger two, which have Arg149 in the +6 a-helical position, bind with UASl mutant sequences having G5 very strongly, T5 strongly, A5 intermediately, and C5 weakly. Mutation of Arg149 to Gln decreased DNA binding to G5 and T5 while increasing its binding to A5 and C5 probes, thus showing the expected Arg149 interaction with G5 or T5 in UASl DNA. ABSTRACT:
The goal of being able to design a zinc-finger transcription (Brown et al., 1985; Miller et al., 1985). A structural model factor which could recognize and bind any given DNA for these Cys2,Hisz zinc fingers (Berg, 1988; Gibson et al., sequence, a critical step in controlling gene expression, has 1988) was subsequently confirmed by 2D NMR' studies of profound consequences. The interaction of a few zinc-finger finger peptides, showing two conserved Cys and two His proteins having only two or three Cysz,His2 fingers (Le., SP1, residues coordinate tetrahedrally with a zinc atom at the base of the finger, while conserved Phe and Leu residues nestle Zif268 (EGRl), Krox20, or ADR1) with their DNA binding sites is relatively well-understood (Berg, 1992; Desjarlais in the hydrophobic core of the finger between a p-turn and & Berg, 1993). However, DNA-binding studies of multiple a-helix (Parraga et al., 1988; Klevit et al., 1990; Lee et al., zinc-finger proteins such as GLI or TFIIIA, with five to nine 1989; Omichinski et al., 1992; Hoffman et al., 1993). X-ray fingers, have revealed complex and unexpected proteincrystallography of the Zif268-DNA complex revealed an DNA interactions between finger amino acid residues and elegant architecture for zinc-finger interaction with DNA which is simple, yet attractive (Pavletich & Pabo, 1991). the bases in bound DNA sequences (Berg, 1992; Pavletich Each finger has three critical amino acids spaced every third & Pabo, 1993; Clemens et al., 1993). residue on the outside of the a-helical domain (at positions The prototype zinc-finger protein, TFIIIA, has nine repeats -1, +3, and +6), which can potentially contact three of about 30 amino acids (--Cys-X2-4-Cys-X3-Phe-X5-Leuconsecutive base pairs in inverted order (3'-5') in the DNA Xz-His-X3-4-His) including a spacer Thr-Gly-Glu-Lys-Pro major groove (see Figure 1). The major protein-DNA interaction, as predicted theoreti'This research was supported by a grant from Research Corp., cally by Seeman et al. (1976), was found to be H-bonding Cottrell College Grant C-3149, generously provided by Bristol-Myers between arginine (at a-helix positions -1 and +6) and Squibb Co. guanine bases (Figure 6A) observed in the Zif268-DNA * To whom correspondence should be addressed. Present address: Ophthalmology Research Laboratories, Cedarsstructure as well as in most zinc-finger proteins studied to Sinai Medical Center Research Institute, Los Angeles, CA 90048. date. Specificity in DNA sequence binding was also 5 Present address: Department of Biochemistry, University of determined by the central fingertip (+3) amino acid histidine California, Riverside, CA 92507. 'I Present address: Department of Immunology, City of Hope H-bonding to guanine or adenine (Figure 6C), as seen in Beckman Research Institute, Duarte, CA 91010. zinc fingers of many proteins including mouse Zif268 and Present address: Department of Plant Pathology, University of related factor Krox20 (Pavletich & Pabo, 1991; Nardelli et California, Davis, CA 95616. # Present address: Department of Biology, University of California, San Diego, CA 92037. * Present address: Department of Molecular and Cellular Biology, University of California, Berkeley, CA, 94720. Abstract published in Advance ACSAbstructs, February 15, 1995. @
I Abbreviations: PCR, polymerase chain reaction; IPTG, isopropyl thio-P-D-galactoside; ,f?Gal, ,f3-galactosidase; NMR, nuclear magnetic resonance; H-bonding, hydrogen bonding; 2D, two dimensional; GEMS, gel electrophoretic mobility shift.
0006-2960/95/0434-3222$09.00/0 0 1995 American Chemical Society
Biochemistry, Vol. 34, No. 10, I995 3223
Designing ADRl Zinc-Finger Mutations al., 1991, 1992), human SP1 and Wilm's tumor suppressor WT1 (Desjarlais & Berg, 1992a,b, 1993; Drummond et al., 1994), and yeast ADRl and MIGl (Thukral et al., 1992; Lundin et al., 1994), but not Tamtrack (Ttk) of Drosophila (Fairall et al., 1992, 1993). These zinc finger (ZF) proteins are shown bound to their cognate DNA elements (3'-5' of top strand), with presumed H-bonds (I) or "non-contacting" specific interactions (!) between critical (-1, +3, and +6 a-helix) amino acids and bases, indicated below. reference
Pavletich/
Desjarlais
Nardel 1 i organism protein Zn finger
ZZl iiU LE2
LE2
E l iiU ?SE i 4
a-helix #
-136-136-136
-136-136-136
-136-136-136-136
aa residue
RER RHT RER
KHA RER RHK
KHM RQR RHT RER
binding
/ ! / / I
11
11
/!Ill1
AN
1 1 1 1
Lundin
organism
yeast
yeast
Dros
protein
ADRl
MIGl
Tamtrafk
Fairall
.
Zn finger
El iiU
ZZl iiU
- Z E l Zp2
a-helix #
-136-136
-136-136
-1236-136
HSNR RNA
aa residue
RHR RLR
RHR RER
binding
Ill I
IIII!I
IIIII
DNA 3 ' - > 5 '
GAG-GTT-5'
GGG-GCG-5'
TAG-GAA-5'
or base(s)
G
cG
I!I
t
At
Thukral
reference
G45'
FIGURE 1: UASl DNA bases interacting with critical ADRl zinc finger residues. Top sequence is UAS 1 top strand DNA bases 4- 11 (in reverse order 3'-5'). Slanted arrows above Tg and T5 indicate all (sixteen) possible changes made in UASl DNA sequence. Lines beneath DNA connect to putative DNA-contacting residues (boxed). Circled residues indicate amino acids homologous to conserved residues of Cys2,His2zinc finger motif.
GGG-GCG-GGG-5' GGG-GCG-GGG-GCG-5'
DNA 3 ' - > 5 ' GCG-GGG-GCG-5'
or base(s)
ets''T5'
et al.
SP1
/!I
GIO A Q Gs G7
human
human
W 6 8 / K r W
Ai1
..
IA. C. G. or T1
Drununond
and Berg
mouse
3'
A
Alternate DNA bases at ambiguous positions (if known from mutation studies) are shown below the DNA sequence, with weaker binding shown in small letters (N is any base). Fidelity in binding of amino acids Arg at -1 or +6 to G, and His at +3 to A or G, is highly specific. Interaction between Glu at $3 and C could be important in specific recognition of cytosine, in which crucial residues of ArgGlu-Arg (RER) bind to a GCG base triplet subsite, as shown for Krox20, SP1, WT1, and MIG1. However, this may be indirect (!), since close Glu-C contact was not seen in the Zif268-DNA complex (Pavletich & Pabo, 1991). Furthermore, Asn at the +3 position of both Tamtrack fingers bound specifically with A, like Gln in Figure 6B (Fairall et al., 1993). Thr, Ala, or Met at +6 in various zinc fingers would tolerate proximity to any of the four DNA bases, with a slight preference for G (Nardelli et al., 1992; Kriwacki et al., 1992). Among the known Cys2,His2 finger proteins, specific binding of thymine has not been adequately studied. The existence of two T's in the DNA binding site for the yeast ADRl protein's second zinc finger provides an opportunity to investigate the specific recognition of thymine. The ADRl transcription factor requires two zinc fingers (Figure 1) (Blumberg et al., 1987), both necessary for activating transcription of mRNA for the glucose-repressible isozyme alcohol dehydrogenase I1 (ADH2), as well as glycerol kinase (GUT1) and catalase A (CTA1) in the yeast Saccharomyces cerevisiae (Denis & Young, 1983; Irani et al., 1987; Pavlik et al., 1993; Simon et al., 1991). Glucose repression of these genes is mediated in part by CAMP-
dependent phosphorylation of ADRl, which inhibits activation of transcription (Cherry et al., 1989), while allowing normal DNA binding of ADRl to the upstream activation site one (UAS1) located at -215 bp in the ADH2 promoter (Taylor & Young, 1990). ADRl protein binds strongly to UAS 1, a 22 bp DNA sequence having dyad symmetry (Eisen et al., 1988). UASl has two inverted 11 bp sites (5'TAAGTTGGAGA-3') containing four critical purine residues (GGAG) that can bind ADRl monomers independently (Thukral et al., 1991a; Cheng et al., 1994). Deletion and mutation studies showed the minimal domain of ADRl required for DNA binding contains the two zinc fingers (amino acids 103-170) plus an additional 20 residues (80102) located amino-terminal to the finger region, involved in phosphate backbone contacts (Thukral et al., 1989; Camier et al., 1992). Within the two zinc fingers of ADR1, alanine-scanning mutagenesis revealed four DNA-binding residues in the "fingertips" including Argll5, Hisll8, and Argl21 (RHR at -1, +3, and +6 a-helical positions) in finger one, and Arg143 (position - 1) in finger two, which were necessary for DNA binding to UAS 1 bases ( ~ ' - T ~ T ~ G , - G ~ A ~ G I O ) (Thukral et al., 1991b, 1993). The conserved Cys, His, and Phe residues were necessary in DNA binding, presumably for zinc coordination and proper folding of each finger (Figure 1, circled residues). Also the two carboxylic amino acids, Glu117 and Asp145, are probably required to coordinate respectively with DNA-binding Argll5 and Arg143 residues located at analogous (- 1) positions within the two zinc fingers, similar to the Asp-supported Arg residues that bind to G in the Zif268-DNA structure (see Figure 6A) (Pavletich & Pabo, 1991). Change-of-specificity mutants were made in ADRl at the four fingertip positions, showing complementation in interaction between mutant ADRl with mutant UAS sequences for binding of Arg115-Gln (at position -1) to AIO, His118-Thr (at +3) to CS, and Arg121-Asn (at +6) to A8 UAS mutant DNA (Thukral et al., 1992). Thus, the three crucial amino acid residues Arg115, His1 18, and Arg121 (RHR) in the first zinc finger are in the correct position to bind respectively to the three bases G10A9G8 (or a GGG mutant) of UASl in inverse order (3'-5') (Figure 1, and shown above), similar to Zif268's zinc finger two residues RHR binding to the GGG sequence.
Taylor et al.
3224 Biochemistry, Vol. 34, No. IO, 1995 A.
A D R l PCRplus “/5/////*
Sphl j EcoRV
Hindlll Sal1 Ncol EcoRI
I
ATG
F1
I
F2
I
(S)
\ -.ik
