The C-Terminal Region of Cytoplasmic Polyadenylation Element Binding Protein Is a ZZ Domain with Potential for Protein–Protein Interactions Daniel J. Merkel, Sarah B. Wells, Bryce C. Hilburn, Fatima Elazzouzi, Gabriela C. Pérez-Alvarado and Brian M. Lee Department of Chemistry and Biochemistry, Southern Illinois University, 1245 Lincoln Drive MC 4409, Carbondale, IL 62901, USA Correspondence to Brian M. Lee: brianlee@siu.edu http://dx.doi.org/10.1016/j.jmb.2013.03.009 Edited by M. F. Summers Abstract Cytoplasmic polyadenylation element binding protein (CPEB) provides temporal and spatial control of protein synthesis required for early development and neuronal synaptic plasticity. CPEB regulates protein expression by inhibiting polyadenylation of selected mRNA transcripts, which prevents binding of the ribosome for protein synthesis. Two RNA recognition motif domains and a C-terminal binuclear zinc-binding domain are required for mRNA binding, but the zinc-binding domain is not required for sequence-specific recognition of the targeted mRNA transcript. The structure and function of the zinc-binding domain of CPEB are unknown. The C-terminal region of CPEB may participate in assembly of the ribonucleoprotein complex that includes the scaffold protein, Symplekin, and the cleavage and polyadenylation specificity factor. Sumoylation of Symplekin is required for polyadenylation, and both cleavage and polyadenylation specificity factor and poly(A) polymerase are sumoylated. The foreshortened poly(A) tail is maintained by poly(A) ribonuclease, which associates with CPEB. While zinc-binding domains are renowned for nucleic acid recognition, binuclear zinc-binding structural motifs, such as LIM (Lin-11, Isl-1, Mec-3), RING (really interesting new gene), PHD (plant homeodomain) and ZZ (ZZ-type zinc finger) domains, participate in protein–protein interactions. Here, we report the solution structure of the C-terminal zinc-binding domain of CPEB1 (CPEB1-ZZ), which has a cross-braced zinc binding topology. The structural similarity to other ZZ domains suggests that the CPEB1-ZZ domain recruits sumoylated proteins during assembly of the ribonucleoprotein complex prior to mRNA export from the nucleus. © 2013 Elsevier Ltd. All rights reserved. Introduction The cytoplasmic polyadenylation element binding protein (CPEB) regulates the expression of proteins required for both early development and synaptic plasticity. 1 Previous studies on oogenesis and em- bryonic development have shown that translational regulation is required to establish polarity leading to cellular differentiation in accordance with the body plan. 2 In Xenopus laevis oocytes, CPEB helps to sequester maternal mRNA in the cytoplasm and prevents the initiation of protein synthesis. 3 Upon activation by phosphorylation, CPEB directs the polyadenylation of the bound mRNA transcript, which induces changes in the 5′ mRNA cap-binding complex leading to the initiation of protein synthesis. 4 Synaptic plasticity requires both temporal and spatial localization of protein synthesis at specific dendrites to modify the synaptic response. 5 The role of CPEB in establishing the synaptic mark has been character- ized in Aplysia californica utilizing the sensory neuron responsible for gill withdrawal reflex to observe long- term facilitation as a model of synaptic plasticity. 6 Four genetically distinct isoforms of CPEB are found in humans with CPEB1 (62.6 kDa) being the closest in homology to the X. laevis (79.4% identity). CPEB1 contains an N-terminal regulatory domain, a PEST (Pro-, Glu-, Ser- and Thr-rich region) motif, 7 two RNA recognition motifs (RRMs) (RRM1 and RRM2) and a Cys 6 -His 2 zinc-binding domain. 8 The mRNA transcripts targeted for regulation by CPEB1 contain a uracil-rich cytoplasmic polyadenylation element (CPE) within the 3′ untranslated region with the consensus sequence UUUUUAU. 9,10 The 0022-2836/$ - see front matter © 2013 Elsevier Ltd. All rights reserved. J. Mol. Biol. (2013) 425, 2015–2026