Metal-dependent Structure and Self Association of the RAG1 Zinc-Binding Domain

ZDD and a fragment of RAG1 including only the RING finger sequence have been expressed in E. coli as fusion proteins with maltose binding protein (MBP). These proteins are referred to as MBP-ZDD and MBP-RF, respectively. We have recently described the cloning, expression, and purification of MBPZDD and MBP-RF. In addition, the proteolytic cleavage of the MBP-ZDD chimera to generate the ZDD fragment, and its subsequent purification, was done as previously reported [5]. N-terminal amino acid sequencing was done at the W.M. Keck Foundation Biotechnology Resource Laboratory, and electrospray mass spectrometry was done by Walter McMurray at the Yale University School of Medicine. The Zn2-coordinated form of ZDD (Zn 2-ZDD) was produced by dialysis of the native ZDD against nitrogen-saturated 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM 2-mercaptoethanol (BME), and 1 mM EDTA at 4˚C for 17 hours. The Zn2-Cd1 form of ZDD was generated by dialysis of Zn2-ZDD against nitrogen-saturated 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM BME supplemented with one molar equivalent of CdCl2 at 4˚C for 12 hours. The metal to protein stoichiometry was determined by atomic absorption spectroscopy using an Instrumentation Laboratory IL157 spectrometer. The concentration of metal ions in the RAG1 proteins was measured in solutions of 3 to 10 µM protein. These were compared to either a Zn or Cd calibration curve ranging from 1 to 15 µM, which was measured prior to each protein sample.
B.
Circular Dichroism Spectroscopy
Circular dichroism (CD) spectra were collected on an AVIV model 62DS spectropolarimeter using a 0.2 mm path length cell. Protein samples were dialyzed against buffer containing 20 mM sodium phosphate (pH 7.0), 50 mM NaCl, 1mM BME. Five separate spectra with a step size of 0.5 nm and a 1.5 nm bandwidth were averaged to obtain the final spectrum for each protein sample. The temperature during the scans was maintained at 25˚C with a water-jacketed cuvette holder. The molar ellipticity was determined using protein concentrations obtained from amino acid analysis. Thermal denaturation studies were done by collecting data at a single wavelength and increasing the temperature in 1˚C increments, equilibrating for 60 sec, and using a 30 sec signal integration time. The melting temperatures (Tm) were determined from the temperature at which the slope of the first derivative of the data was at a minimum.
I. Introduction
Structural zinc-binding domains are often characterized by the requirement of zinc coordination for proper protein folding [1]. One specific class of zinc-binding motif that will be discussed here is the zinc C3HC4 motif, also known as the RING finger [2]. To date at least eighty proteins include a sequence of approximately 50 residues consistent with a RING finger motif. This conserved sequence, with minor variations in some cases, is defined as follows: C-X2-C-loopI-C-X-H-X2-C-X2-C-loopII-C-X 2-C, where X represents any amino acid. A common function attributable to the RING finger module has remained elusive, although a role in protein-protein interactions has been speculated [2]. One of the first RING finger sequences was identified in RAG1, a protein expressed in developing lymphocytes by recombination activating gene-1 [3]. RAG1, along with RAG2, is an essential component of the V(D)J recombination reaction, which produces the genetic sequence encoding for the variable regions of the T cell receptor and immunoglobulin chains. Briefly, V(D)J recombination is accomplished via selection and assembly of gene segments known as variable (V), joining (J), and sometimes diversity (D) in an ordered and precisely regulated process (for a review see [4]). The RING finger sequence of RAG1 is present within the N-terminal third of the protein, which contains a total of 1040 residues in the murine form. Besides the RING finger sequence, we have recently identified the presence of two C2H2 zinc finger sequences within RAG1 [5]. A domain in RAG1 containing one of the zinc finger modules plus the RING finger forms a highly specific dimer, as characterized by a variety of biophysical techniques [5]. The dimerization of this zinc-binding domain provides further support for the participation of RING fingers in protein-protein interactions. This dimerization domain of RAG1, previously referred to as R121, will be referred to here as ZDD, zinc-binding dimerization domain. Here we focus on the role of metal binding to the ZDD dimer. In particular, we have investigated the stabilities of different species of ZDD with varying metal-to-protein stoichiometries. Combined with the metal-binding studies, we have further investigated dimer