- Top of page
- Materials and methods
The Yeast MATa1 and MATα2 are homeodomain proteins that bind DNA cooperatively to repress transcription of cell type specific genes. The DNA affinity and specificity of MATa1 in the absence of MATα2, however, is very low. MATa1 is converted to a higher affinity DNA-binding protein by its interaction with the C-terminal tail of MATα2. To understand why MATa1 binds DNA weakly by itself, and how the MATα2 tail affects the affinity of MATa1 for DNA, we determined the crystal structure of a maltose-binding protein (MBP)-a1 chimera whose DNA binding behavior is similar to MATa1. The overall MATa1 conformation in the MBP-a1 structure, which was determined in the absence of α2 and DNA, is similar to that in the a1/α2/DNA structure. The sole difference is in the C-terminal portion of the DNA recognition helix of MATa1, which is flexible in the present structure. However, these residues are not in a location likely to be affected by binding of the MATα2 tail. The results argue against conformational changes in a1 induced by the tail of MATα2, suggesting instead that the MATα2 tail energetically couples the DNA binding of MATα2 and MATa1.
The MATa1 protein (which we shall call a1), plays an important role in yeast mating-type regulation (Johnson 1995). In diploid a/α cells, a1 binds DNA cooperatively with MATα2 (which we shall call α2), leading to the repression of haploid-specific genes (hsg). Previous genetic, biochemical, and structural studies have characterized the minimum functional fragments of both proteins necessary for DNA binding and heterodimerization (Goutte and Johnson 1993; Phillips et al. 1994; Li et al. 1995; Vershon et al. 1995). The α2 protein binds DNA with a homeodomain located near the carboxyl-terminus of the protein and contacts the a1 homeodomain with an 18-amino acid carboxyl-terminal tail that is unstructured in the absence of a1 (Wolberger et al. 1991; Li et al. 1995). The a1 protein, which also contains a homeodomain at its carboxyl-terminus portion, shows no reproducible sequence-specific DNA binding. However, in combination with α2, the a1/α2 heterodimer binds DNA with high affinity, and the sequence specificity of the heterodimer increases 3000-fold over that of α2 alone (Goutte and Johnson 1993; Phillips et al. 1994).
Structures of the a1/α2/DNA and the α2/DNA complex have been determined by X-ray crystallography (Wolberger et al. 1991; Li et al. 1995). In the a1/α2/DNA structure, the a1 and α2 homeodomains bind DNA in a head-to-tail orientation, with heterodimer contacts mediated by the 18-residue carboxyl-terminal tail of α2 (Fig. 1). The α2 tail becomes ordered only in the presence of a1, forming a short amphipathic helix that packs against the a1 homeodomain between helices 1 and 2. An evenly distributed 60° bend in the DNA is induced by the binding of the a1/α2 heterodimer. The conformation of α2 and its DNA contacts are virtually the same in both the a1/α2/DNA and the α2/DNA structures. The docking of the a1 homeodomain on the DNA is similar to that of the α2 protein. a1 contacts DNA bases in the major groove with five residues in helix 3: Val47, Ile50, Asn51, Met54, and Arg55 (Li et al. 1995). Unlike many other homeodomain proteins, the N-terminal arm of a1 is mostly unstructured, does not contact the DNA minor groove, and was shown to be dispensable for DNA binding (M. Stark and A.D. Johnson, pers. comm.).
Given the similar docking of a1 and α2 to DNA and the extensive a1-DNA contacts, the prior structures have not explained why a1 binds DNA so weakly in the absence of α2. Also unclear is how the binding of the α2 tail to the non-DNA binding face of a1 dramatically increases the DNA binding affinity and specificity of a1. Two hypotheses have been proposed concerning the role of the α2 tail: (1) the α2 tail couples the DNA binding of a1 and α2, making the binding of a1 to DNA energetically more favorable than it would be in the absence of cooperativity; (2) upon heterodimarization, contacts with the α2 tail causes a1 to bind DNA with higher affinity and specificity. In support of the latter hypothesis, Stark et al. (1999) demonstrated that when the α2 tail is covalently linked to the homeodomain of a1, the engineered a1 can bind DNA tightly and specifically as a monomer (Stark et al. 1999). They also showed that an α2 peptide supplied in trans can induce tighter DNA binding by the a1 homeodomain. NMR studies of the free a1 protein further suggested that the α2 tail induces changes in the loop 1 region between helices 1 and 2 of a1 that push it towards a properly folded DNA binding conformation (Anderson et al. 2000).
To understand fully why a1 binds DNA so weakly, and how binding of the α2 tail to the a1 homeodomain improves the affinity of a1 for DNA, we set out to determine the crystal structure of the a1 homeodomain in the absence of DNA or the α2 protein. To overcome our inability to obtain crystals of free a1, we took an alternative approach and determined the structure of a chimeric protein consisting of the free a1 homeodomain fused to the maltose binding protein (MBP). The DNA binding properties of the MBP-a1 chimera are identical to that of the a1 homeodomain. The 2.1-Å crystal structure shows that the C-terminal portion of the DNA recognition helix of a1 is unstructured in the absence of the α2 tail and DNA, providing an explanation for the weak affinity of a1 alone for DNA. Other than the four C-terminal residues, the structure of the a1 homeodomain in the MBP-a1 structure is identical to that in the a1/α2/DNA structures. The absence of a conformational change leads us to conclude that the α2 tail probably increases the DNA specificity and affinity of a1 by coupling the DNA binding of a1 and α2 to minimize the overall energy cost. We discuss the possible sources of discrepancies between our crystal structures and the previous NMR structure.
- Top of page
- Materials and methods
The reason the a1 homeodomain contributes high DNA binding affinity and specificity to the a1/α2/DNA complex, while having little intrinsic affinity and specificity for DNA in the absence of α2, has remained an open question. Previous results (Stark et al. 1999) had suggested that the tail of α2, which mediates all protein–protein contacts with a1 in the a1/α2/DNA structure, may play an allosteric role in inducing a1 to adopt a high-affinity DNA-binding conformation. A report (Anderson et al. 2000) comparing the solution NMR structure of the free a1 homeodomain with the (Anderson et al. 2000) crystal structure of a1 in a ternary complex with α2 and DNA (Li et al. 1995) identified structural differences located in the loop 1 region connecting helices 1 and 2 and in the C-terminus of a1. The authors proposed that changes in loop 1 of a1 induced by the α2 tail cause van der Waals stacking changes leading to the ordering of a final turn in the DNA-binding helix of a1. We took a different approach by determining the crystal structure of the free a1 homeodomain. The 2.1- and 2.3-Å resolution crystal structures from two crystal forms allow us to observe crystal structure of the free a1 and to compare bound and free structures determined by the same experimental technique. The fused MBP in the present crystal structure is unlikely to have altered the a1 conformation, because the DNA binding properties of the fusion protein are the same as the a1 homeodomain, and MBP does not contact the α2 tail binding site on a1. Interestingly, we draw different conclusions from our study.
The comparison of bound and free crystal structure of a1 suggests that the flexibility of the C-terminus of the a1 recognition helix can explain why the a1 homeodomain binds DNA weakly in the absence of α2. The free a1 conformation determined from the MBP-a1 structures clearly shows that the carboxyl-terminal portion of the DNA recognition helix (helix3) of a1 is destabilized in the absence of α2 and DNA. The conformation of two important DNA recognition amino acid residues is completely different from their DNA-bound conformation, and would occlude DNA binding unless they underwent a conformational change docked onto DNA. This part of our observation is in agreement with that from the solution NMR structure of the a1 homeodomain suggesting a poorly folded a1 C-terminus (Phillips et al. 1991). We note that an unstructured C-terminus of helix 3 has been found in several other free homeodomain protein structures. The last four residues of the free engrailed homeodomain crystal structure are disordered (Clarke et al. 1994), as are the last 10 and 8 residues of the free VND-NK2 and Antp, respectively (Billeter et al. 1990; Tsao et al. 1995). However, the other homeodomain proteins with an unstable helix 3 C-terminus retain a functional N-terminal arm that makes DNA minor groove contacts. Because the N-terminal arm of a1 does not appear to participate in DNA binding, the requirement for reordering the C-terminal residues of helix 3 may have proportionally greater effect on the affinity of a1 for DNA.
We did not observe any conformational differences in the remainder of the a1 homeodomain that would suggest an allosteric role for the binding of the α2 tail. This observation disagrees with the conclusion from the NMR structural analysis (Baxter et al. 1994; Anderson et al. 2000), which links van der Waals stacking changes in the a1 loop 1 region caused by the binding of the α2 tail to the ordering of a final turn in the DNA-binding helix. There are several discrepancies between the two studies. The r.m.s.d. of Cα positions (residues 10–52, excluding the flexible N- and C-terminus) in a superposition of the NMR structure of free a1 and the crystal structure of DNA-bound a1 is 1.83Å (Anderson et al. 2000), significantly higher than the 0.4 Å r.m.s.d. we observe comparing the crystal structures of the free and DNA-bound a1 protein. Most of the side-chain conformation changes in the loop 1 region observed in the NMR study are not supported by our crystal structure. Moreover, the relative orientations of the three homeodomain helices remain the same in the crystal structures of the free and DNA-bound a1. The apparent discrepancy is unlikely due to differences in experimental conditions such as pH and temperature at which the NMR and X-ray diffraction data were collected. We note that the free a1 conformation does not change when the MBP-a1 crystals are transferred from pH 5.0 to pH 8.0 (data not shown). The room temperature NMR structure is expected to be more dynamic than the crystal structures determined at 90 K, but this is unlikely to account for a significant part of the discrepancy in the Cα alignments.
We think it most likely that the apparent discrepancy between the two studies is the result of the different structure determination methods used. The conclusions of the previous study relied upon the assumption that differences between the NMR structure of free a1 and the crystal structure of bound a1 were attributable to the effects of binding to α2 and DNA. However, it has previously been observed that structures of a given protein determined by NMR and crystallographic methods do not necessarily superimpose well. For example, when the core Antennapedia homeodomain (residues 5–60) in the crystal structure of Antennapedia homeodomain–DNA complex are aligned with the 16 NMR models of the same complex, the average r.m.s.d. in Cα positions is 1.13 Å ( Billeter et al. 1993; Qian et al. 1993; Fraenkel and Pabo 1998). In the case of Ets-1 bound to DNA, the NMR (Werner et al. 1997) and crystal structures (Garvie et al. 2001) of the Ets domain superimpose with an r.m.s.d. of 2.3 Å. The previously observed discrepancies are of the same order of magnitude as the differences between the NMR and crystal structures of a1. Moreover, we note that the Ramachandran plot of a randomly picked free a1 NMR structure (Anderson et al. 2000) out of 20 in the ensemble has only 67% of the amino acid residues in the most favored regions, with one residue (2%) in the disallowed region. In contrast, the crystal structures we report here have 96% of the residues in the most favored regions and no residues in the disallowed or generously allowed regions. We also note that the NMR structure determination of a1 (Anderson et al. 2000) did not include measurements of hydrogen-bonding patterns (Grzesiek et al. 2001), or distance-independent residual dipolar coupling (Tjandra and Bax 1997), which have the potential to reduce the discrepancy between NMR and crystal structures. Although none of the aforementioned caveats rule out the possibility of a global conformational change, these considerations lead us to favor a model in which conformational changes are likely to be due to DNA binding alone and are localized to the C-terminus of the a1 homeodomain helix 3.
Because α2-induced conformational changes in a1 are unlikely according to our data, we instead favor an energetic coupling model, in which the DNA binding of a1 and α2 are strictly coupled to the heterodimerization of a1 and α2 mediated by the α2 tail. The a1 protein cannot dissociate from DNA without breaking the heterodimer contacts with the α2 protein. In such circumstances, the dissociation constant of the a1/α2 heterodimer will be the product of the dissociation constant of the two individual proteins. The dissociation constant of a1 from DNA is estimated to be from 10−5 to 10−6 M and that of α2 is ∼10−8 M. The strict coupling model predicts a 10−13 to 10−14 M2 dissociation constant of the a1/α2 heterodimer from DNA, which is exactly what was observed in the DNA-binding assays. Besides, the heterodimerization of a1/α2 obviously provides the free energy to compensate for the folding of the a1 C-terminus, which occurs upon binding to DNA. The seemingly weak Kd of the heterodimerization of a1 and α2 in solution, estimated to be 2 × 10−4 M (Phillips et al. 1994), is expected to be tighter in the a1/α2/DNA ternary complex, because the nearby DNA and a1 molecules further restrict the conformational space of the α2 tail, dramatically reducing the entropic cost of folding the α2 tail during heterodimerization. The above rationales, rather than an allosteric mechanism, are the more likely explanations for the role of the α2 tail in recruiting a weakly binding partner, a1, to the DNA.