NMR structure of the hRap1 Myb motif reveals a canonical three-helix bundle lacking the positive surface charge typical of Myb DNA-binding domains

Mammalian telomeres are composed of long tandem arrays of double-stranded t elomeric TTAGGG repeats associated with the telomeric DNA-binding proteins, TRF1 and TRF2. TRF1 and TRF2 contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In the budding yeast, telomeric DNA is associated with scRap1p, which has a central DNA-binding d omain that contains two structurally related Myb domains connected by a lon g linker, an N-terminal BRCT domain, and a C-terminal RCT domain. Recently, the human ortholog of scRap1p (hRap1) was identified and shown to contain a BRCT domain and an RCT domain similar to scRap1p. However, hRap1 containe d only one recognizable Myb motif in the center of the protein. Furthermore , while scRap1p binds telomeric DNA directly, hRap1 has no DNA-binding abil ity. Instead, hRap1 is tethered to telomeres by TRF2. Here, we have determi ned the solution structure of the Myb domain of hRap1 by NMR. It contains t hree helices maintained by a hydrophobic core. The architecture of the hRap 1 Myb domain is very close to that of each of the Myb domains from TRF1, sc Rap1p and c-Myb. However, the electrostatic potential surface of the hRap1 Myb domain is distinguished from that of the other Myb domains. Each of the minimal DNA-binding domains, containing one Myb domain in TRF1 and two Myb domains in scRap1p and c-Myb, exhibits a positively charged broad surface that contacts closely the negatively charged backbone of DNA. By contrast, the hRap1 Myb domain shows no distinct positive surface, explaining its lac k of DNA-binding activity. The hRap1 Myb domain may be a member of a second class of Myb motifs that lacks DNA-binding activity but may interact inste ad with other proteins. Other possible members of this class are the c-Myb R1 Myb domain and the Myb domains of ADA2 and Adf1. Thus, while the folds o f all Myb domains resemble each other closely, the function of each Myb dom ain depends on the amino acid residues that are located on the surface of e ach protein. (C) 2001 Academic Press.