The MADS box family of transcription regulatory proteins contain highly conserved DNA-binding domains. The proteins in this family bind to conserved DNA sites called CArG boxes, and many of the MADS box proteins can bind to each other's sites with good affinity in vitro (37
). This raises the question of whether these proteins can function for each other in vivo and, if not, what determines their specific differences. To address this question, we examined the specificity and role of the Arg80 MADS box protein in the regulation of the transcription of arginine-metabolic genes in S. cerevisiae
. In addition to Arg80, there are three other MADS box proteins in S. cerevisiae
, Mcm1, Smpl, and Rlm1. We have shown that Rlm1 and Smp1 do not appear to be involved in the regulation of arginine-metabolic genes. We have also shown that although the U. maydis
Umc1 protein can partially replace Mcm1 in the regulation of arginine, neither it nor Mcm1 can fulfill the role of Arg80. These results show that the few differences between the MADS box sequences of the Arg80 and Mcm1 proteins are sufficient to determine the specificity of the Arg80 protein.
The MADS box domains of the Arg80 and Mcm1 proteins are very similar in their sequences and are likely folded into similar structures. We have therefore used the crystal structure of Mcm1 as a model for understanding how Arg80 may fold and bind DNA (38
). Many of the residues involved in DNA contacts in Mcm1 are conserved in Arg80, so it is also likely that these proteins bind DNA in a similar manner (Fig. ). However, we have shown that the substitution of the Mcm1 MADS box domain into the context of Arg80 does not complement an arg80
mutant, indicating that there are significant differences in the functional specificities of the two proteins. It is likely that a significant part of this difference in specificities is due to differences in interactions with the cofactors that bind to Arg80. Amino acids at positions that are not conserved between the two proteins most likely determine the specificity of the interactions of Arg80 with Mcm1 and Arg81. To identify which regions of the Arg80 MADS box domain specify these interactions, we replaced residues in Arg80 with amino acids found at the same relative positions in the Mcm1 MADS box domain and examined which substitutions have an effect on Arg80 function. We also performed the converse experiment to determine whether substitutions in the Mcm1 MADS box domain would change its specificity to that of Arg80. Our mutational analysis shows that while substitutions in any one region of the protein have little effect on their own, in combination with mutations in other regions of the protein they significantly affect Arg80-dependent regulation. The fact that mutations in any one region do not affect activity on their own suggests that the remaining interactions with the other regions of the protein are able to compensate for the loss of proper contacts by the mutated residues. This result suggests that there are multiple points of interaction between Arg80 and its cofactors and that the interface between Arg80 and its cofactors may be very extensive.
In the initial mutagenesis of Arg80, multiple amino acid substitutions were introduced within each secondary-structure region of the MADS box domain. In combination with mutations in other regions of the MADS box domain, these changes caused a large decrease in activity. However, these substitutions could have multiple effects on the activity of the protein by, for example, altering DNA-binding affinity, stability, protein folding, or protein interactions. Therefore, to investigate which residues within a region are important for the interaction with the Arg80 cofactors, we made individual substitutions within a region. For example, although other substitutions in the N-terminal arm alter the DNA-binding properties of the protein, we have shown that only the conservative phenylalanine substitution at position Y87, which does not alter DNA-binding activity, affects the regulatory function of the protein. We have also shown that substitutions at residues Y110 and T135 cause a significant decrease in Arg80 activity. The fact that we can switch the specificity of the Mcm1 MADS box domain to that of Arg80 by swapping the amino acids at these positions in combination with residues in the βI strand indicates that these are important determinants of the specificity of the protein. The observation that the Y110F T135S (pAJ240) and Y87F Y110F (pAJ234) double mutants and the Y110F, A118T, N119Q (pAJ254) triple mutant have such a large effect on Arg80 function further supports the notion that these residues play an important role in the function of the protein.
Although we found that the residues that define Arg80 specificity lie in several different secondary-structure regions of the protein (N-terminal arm, αI, βI, and βII), in spatial terms these substitutions all cluster on one side of the protein (Fig. ). This region is located away from the DNA and lies along the edge of the interface between the monomers. Residues from both monomers make up this partially contiguous surface. It is likely that these residues form a surface of the protein that is involved in the protein's interaction with its cofactors. In support of this idea, we have shown that even though some of the swaps increase the DNA-binding affinity of the protein alone to the P site, the protein shows decreased transcriptional regulation in vivo and fails to bind cooperatively in a complex with Arg81 and Mcm1 in vitro (data not shown). This result suggests that these substitutions affect the ability of Arg80 to interact with its cofactors.
FIG. 7. Position of the residues required for Arg80 activity. (A) The relative positions of residues that affect Arg80 function are displayed on a model of the Mcm1 MADS box domain dimer (spacefill) bound to DNA (stick-like figures). One monomer is shown in white, (more ...)
Our results suggest that residues Y87, Y110, A118, N119, and T135 of Arg80 define a partially contiguous surface on the side of the protein that may interact with the protein's cofactors (Fig. ). However, it is likely that many of the conserved residues in this region of the protein also play a role in the interaction of Arg80 with its cofactors. In support of this notion, we have shown that replacement of residue V114, which lies on the surface between Y87 and Y110, may provide a contiguous surface for contacts with an Arg80 cofactor, which is most likely Mcm1. Many of the side chains in this region of the protein are relatively hydrophobic, suggesting that the interactions with the cofactor may be hydrophobic in nature. However, the phenylalanine substitutions at positions Y87 and Y110 significantly decrease the level of Arg80 activity, indicating that the hydroxyl group of the tyrosine side chain at both of these positions plays an important role in the function of the protein. It is possible that these hydroxyl groups are involved in making hydrogen bond contacts to the Arg80 cofactor and contribute to the specificity of the protein interactions.
The mutational analysis indicates that there are several surfaces of the Arg80 protein that interact with its cofactors. The results of a previous mutational analysis suggested that Arg82 interacts with conserved solvent-exposed residues in the αI helix of the Arg80 MADS box domain (14
). We propose that another region for cofactor interactions on the surface of Arg80 is on the side of the protein, i.e., at the dimer interface. This region of interaction is significantly different from the region of Mcm1 that interacts with the yeast MATα2 protein or the surface of the mammalian SRF protein that interacts with SAP-1 (Fig. ) (17
). In both of these cases, the interaction surface is along the hydrophobic groove formed between the βII strand and the αII helix. The regions of MATα2 and SAP-1 that interact with the Mcm1 and SRF MADS box domains, respectively, are also similar to each other and contain an extended strand that lies in the groove. One of the main features of these interactions is a conserved aromatic group that fits into a pocket formed by amino acid side chains corresponding to residues V131, V143, K149, and I152 of Arg80. Amino acid substitutions in this region of Mcm1 affect the interaction with α2 (7
). We have found that substitutions in this region of Arg80 also cause a decrease in activity, suggesting that this region may be involved in contacts with cofactors in a manner similar to the interactions between Mcm1 and α2 and between SRF and SAP1. However, the effects of these mutations are less severe than those of substitutions in other regions of the protein. For example, although the αII swap in Arg80 changes a number of the residues in or around this pocket, the effects of these multiple changes on Arg80 function are relatively weak. In contrast to substitutions in the hydrophobic pocket, the replacement of residues Y110, A118, N119, I120, I124, L125, A126, N127, S128, and T135 has a large effect on Arg80 function. This result differs significantly from that obtained when substitutions are made at the corresponding positions in Mcm1, which do not affect the interaction with α2 (J. Mead and A. K. Vershon, unpublished data).
Our results are consistent with the idea that there are at least two different regions on the Arg80 MADS box domain that can be involved in interactions with cofactors: one formed by the hydrophobic pocket on the face of the protein and the other in a groove across the dimer interface on the side of the protein (Fig. ). Some residues, such as Y110, F134, T135, and P137, may be involved in protein interactions in both regions of the domain. The amino acid side chains of these residues in Mcm1 and SRF are in close contact with both α2 and SAP-1 cofactors and are also important for the interactions of Arg80 with its cofactors. It is possible that if a cofactor binds to one face of the protein and contacts these residues, it may prevent a different cofactor that requires contacts with these residues from binding and force it to bind to the other face of the MADS box domain. Alternatively, it is possible that these two interfaces may simultaneously provide contacts for two different cofactors, helping to form a complex around the MADS box protein. DNA-binding assays showed that Arg80 binds in a complex with Mcm1 and Arg81, and two-hybrid studies showed that Arg80 interacts with Mcm1, Arg81, and Arg82 (4
). The multiple surfaces defined through the mutational analysis of Arg80 may provide the interfaces for interactions with these different cofactors. It is possible that other MADS box proteins use this region to interact with their cofactors and that variations at these residues among the different proteins of the MADS box family may serve to specify the interactions of these proteins with their cofactors.