STRUCTURE NOTE Crystal Structure of Homo sapiens Protein hp14.5 Babu A. Manjasetty, 1,2† Heinrich Delbru ¨ ck, 3,4 Dinh-Trung Pham, 4,5 Uwe Mueller, 1,3 Martin Fieber-Erdmann, 1,3 Christoph Scheich, 4,6 Volker Sievert, 4,6 Konrad Bu ¨ ssow, 4,6 Frank H. Neisen, 4,7 Wilhelm Weihofen, 3 Bernhard Loll, 3 Wolfram Saenger, 3 and Udo Heinemann 2,3 * 1 Protein Structure Factory, c/o BESSY GmbH, Berlin, Germany 2 Forschungsgruppe Kristallographie, Max-Delbru ¨ ck-Centrum fu ¨ r Molekulare Medizin, Berlin, Germany 3 Institut fu ¨r Chemie/Kristallographie, Freie Universita ¨ t Berlin, Germany 4 Protein Structure Factory, Berlin, Germany 5 Alpha-Bioverfahrenstechnik GmbH, Kleinmachnow, Germany 6 Max-Planck-Institut fu ¨ r Molekulare Genetik, Berlin, Germany 7 Universita ¨ tsklinikum Charite ´, Institut fu ¨ r Medizinische Physik & Biophysik Berlin, Germany Introduction. Human trichloroacetic acid–soluble pro- tein p14.5 (hp14.5) is a member of the large YjgF/YER057c/ UK114 protein family, which comprises approximately 200 members, 1 most with unclear biological function. The hp14.5 homologs belonging to the high-identity (38%) group (approximately 80 proteins) share 9 completely invariant residues that are spread throughout the se- quence. 1 The crystal structures of 3 distant homologs have been determined: YabJ from Bacillus subtilis (45% iden- tity), 2 YjgF from Escherichia coli (46% identity), 3 and Yeo7 from yeast (39% identity). 4 These proteins share similar homotrimeric structures and distinct clefts located be- tween the subunits that may be of functional relevance. Recently, the NMR structure of the homologous H10719 from Haemophilus influenzae (44% identity) has been determined. 1 Based on this structure, an NMR ligand screening was performed using a set of molecules related to the various cellular functions reported in the literature (i.e., translation inhibition, 5,6 ribonuclease activity, 7 and involvement in the regulation of purine 8 and isoleucine 9 biosynthetic pathways). Human p14.5 was first isolated from mononuclear phago- cytes. 6 Using a rabbit reticulocyte lysate system, it was shown that recombinant hp14.5 in micromolar concentra- tions and native hp14.5 in nanomolar concentrations inhibit protein synthesis in vivo. Whereas the hp14.5 mRNA is weakly expressed in freshly isolated monocytes, it is upregulated when these monocytes differentiate to macrophages. 6 Clear evidence for a differentiation-depen- dent regulation of hp14.5 synthesis is provided by the observation that in a variety of liver and kidney tumor cells, only a low expression level is observed, whereas in fully differentiated cells, a high level is measured. 6 Inhibi- tion of protein synthesis in vitro, similar to that observed for hp14.5, was described for the homologous rat perchloric acid–soluble protein (L-PSP). 5 The translation-inhibitory activity of L-PSP has been attributed to its endoribonucleo- lytic activity toward single-stranded RNA, 7 which induces disaggregation of the reticulocyte polysomes into 80S ribosomes even in the presence of cycloheximide. Results. Like several homologous YjgF/YER057c/UK114 protein family members, the human translational inhibi- tor protein, hp14.5, adopts a chorismate mutase-like 3,10 subunit fold. Each monomer consists of one globular domain formed by a 6-stranded -sheet and two -helices arranged in roughly parallel orientation on one side of the sheet [Fig. 1(a)]. In the -sheet, the order of strands from one edge to the other is 1-2-3-6-4-5, where 4 and 5 are in parallel and all other strands are antiparallel. Three hp14.5 monomers associate into a trimer with threefold noncrystallographic symmetry [Fig. 1(b)]. Three such trimers formed by 9 polypeptide chains constitute the asymmetric unit of the crystal. Pairwise superpositions of the 3 trimers yield root-mean-square deviation (RMSD) values of 0.399 A ˚ , 0.607 A ˚ , and 0.556 A ˚ , indicating closely related trimer organization. The shape of the trimer resembles a triangular barrel. Three pairs of -helices are positioned on its outer surface, and 3  4 antiparallel strands form the inner surface of the barrel. The barrel is sealed off at one end by Tyr 110 from each of the 3 subunits, and at the other end by 3 other tyrosines, Tyr 32 . The sealing of the barrel leads to the formation of a large cavity filled with water molecules. The biological role of this cavity, if any, is not clear. On the outer surface of the Grant sponsor: German Federal Ministry for Education and Re- search (BMBF) through the “Leitprojektverbund Proteinstrukturfab- rik.” Grant sponsor: Fonds der Chemischen Industrie to Udo Heine- mann and Wolfram Saenger. *Correspondence to: Udo Heinemann, Max Delbru ¨ ck Center for Molecular Medicine, Robert-Ro ¨ssle-Str. 10, D-13092, Berlin, Ger- many. E-mail: heinemann@mdc-berlin.de †On leave from the Department of Physics, Government Science College, Bangalore, India. Received 13 August 2003; Accepted 14 August 2003 PROTEINS: Structure, Function, and Bioinformatics 54:797– 800 (2004) © 2004 WILEY-LISS, INC.