C OMMUNICATION Solution Structure of the MID1 B-box2 CHC(D/C)C 2 H 2 Zinc-binding Domain: Insights into an Evolutionarily Conserved RING Fold Michael A. Massiah 1 ⁎, Jessica A. B. Matts 1 †, Kieran M. Short 2 † Brandi N. Simmons 1 , Suryaparkash Singireddy 1 , Zou Yi 2 and Timothy C. Cox 2,3 1 Department of Biochemistry and Molecular Biology , Oklahoma State University, Stillwater, OK 74078, USA 2 Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria, Australia 3800 3 Division of Craniofacial Medicine, Department of Pediatrics, University of Washington, Seattle, WA 98195, USA The B-box type 2 domain is a prominent feature of a large and growing family of RING, B-box, coiled-coil (RBCC) domain-containing proteins and is also present in more than 1500 additional proteins. Most proteins usually contain a single B-box2 domain, although some proteins contain tandem domains consisting of both type 1 and type 2 B-boxes, which actually share little sequence similarity. Recently, we determined the solution structure of B-box1 from MID1, a putative E3 ubiquitin ligase that is mutated in X-linked Opitz G/BBB syndrome, and showed that it adopted a ββα RING-like fold. Here, we report the tertiary structure of the B-box2 (CHC(D/C)C 2 H 2 ) domain from MID1 using multidimensional NMR spectroscopy. This MID1 B-box2 domain consists of a short α-helix and a structured loop with two short anti-parallel β-strands and adopts a tertiary structure similar to the B- box1 and RING structures, even though there is minimal primary sequence similarity between these domains. By mutagenesis, ESI-FTICR and ICP mass spectrometry, we show that the B-box2 domain coordinates two zinc atoms with a ‘cross-brace’ pattern: one by Cys175, His178, Cys195 and Cys198 and the other by Cys187, Asp190, His204, and His207. Interestingly, this is the first case that an aspartic acid is involved in zinc atom coordination in a zinc-finger domain, although aspartic acid has been shown to coordinate non-catalytic zinc in matrix metalloproteinases. In addition, the finding of a Cys195Phe substitution identified in a patient with X-linked Opitz GBBB syndrome supports the importance of proper zinc coordination for the function of the MID1 B-box2 domain. Notably, however, our structure differs from the only other published B-box2 structure, that from XNF7, which was shown to coordinate one zinc atom. Finally, the similarity in tertiary structures of the B-box2, B-box1 and RING domains suggests these domains have evolved from a common ancestor. © 2007 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: midline-1; B-box2; TRIM/RBCC; RING; NMR † J.A.B.M. and K.M.S. contributed equally to this work. Abbreviations used: GST, glutathione-S-transferase; ESI-FTICR MS, electrospray ionization-Fourier transform ion cyclotron resonance mass spectroscopy; ICP-MS, inductively coupled plasma mass spectrometry; HSQC, heteronuclear single quantum coherence; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MATH, meprin and TRAF-C homology; MID1, Midline-1; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; PHD, p homology domain; PP2Ac, catalytic subunit of protein phosphatase 2A, RBCC, RING-B-box-coiled coil; RING, really interesting new gene; TRIM, tripartite motif; XNF7, Xenopus nuclear factor 7; RMSD, root-mean-square deviation. E-mail address of the corresponding author: massiah@biochem.okstate.edu doi:10.1016/j.jmb.2007.03.017 J. Mol. Biol. (2007) 369,1–10 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.