Abstract Human ubiquitin-like SUMO-1/2/3 proteins have been identified. The 3-D structure of the SUMO-1 has been shown to be very similar to that of ubiquitin, although their sequences share only 18 % identity. Unlike ubiquitination targets proteins for degradation, sumoylation appears to regulate a number of cellular processes such as protein-protein interaction, subcellular localization, protein stability, apoptosis, cell cycle and so on . Our laboratory has cloned cDNAs encoding human SUMO-2, mouse SUMO-2 and SUMO-3, as well as a single SUMO gene from nematode and Drosophila. Recently (Su & Li, Gene 296:65-73,2002), Su & Li have performed data-mining on current human genomic sequence and found the presence of only three SUMO-1/2/3 functional genes located at chromosome no. 2q33, 17q25.1 and 21q22.3, respectively, as well as eight SUMO-1 pseudogenes and 23 SUMO-2 pseudogenes. The protein-coding sequence of SUMO-1 gene is interrupted by four introns , while those of SUMO-2/3 genes are interrupted by only three introns. In this study , most of SUMO-1/2/3 proteins were show to be localized on nuclear membrane, nuclear bodies and cytoplasm, respectively. The N-terminus-deleted SUMO-1 proteins was further shown to be localized on nuclear membrane and in cytosol, while the mutant SUMO-2/3 proteins were localized only in the cytosol. The inactive precursor form of SUMO-3 was exclusively localized in the cytosol. The activation of SUMO-3 in HeLa cells was triggered by actinomycin D and its location was shifted from cytosol to nucleus. Further, the inactive precursor of SUMO-3 was reduced in HeLa cells treated with nocodazole and arsenic trioxide.

The SUMO protein was named Small Ubiquitin-like MOdifier because its 3-D structure was similar to Ubiquitin. In human, three SUMO proteins were discovered, namely, SUMO-1/2/3. The recombinant ¡µ1-8, 94-95 SUMO-2 protein with 10 histidine residues at its N-terminus was expressed using E. coli. BL-21(DE3), purified at 4 oC and crystallized at room temperature. The surface properties of human SUMO-1/2/3 proteins and 3-D structure of ¡µ1-8, 94-95 SUMO-2 protein were analyzed using computer modeling and X-ray diffraction technology respectively. The two-step purification by immobilized metal ion affinity chromatography(IMAC) was developed to yield ¡µ1-8, 94-95 SUMO-2 protein that reached 60 mg/ml for crystallization. On protein expression, 120 mg protein was obtained from 6 L bacterial growth broth. Crystals of ¡µ1-8, 94-95 SUMO-2 were obtained by the hanging-drop vapor diffusion method and many different crystal forms were observed. One of single crystal with triangular plate polyhedron form diffracted to 1.6 Å resolution, the other one with rectangular polyhedron form diffracted to 1.2 Å. Analysis of the diffraction pattern suggests the crystals belong to R3 space group, the former one owned unit cell parameters a= b=75.3 Å, c=29.2 Å, £\=90¢X, £]=90¢X,£^=120¢X, and the later one owned unit cell parameters a= b=74.9 Å, c=33.2 Å and the same angles respectively. The R factor and Rfree of refinement are 0.133 and 0.190 with highly precise phase on 3-D structure of SUMO-2 protein. Comparison of crystal structure between human SUMO-2 and yeast SMT3 showed that the r.m.s. deviation of C£\ coordinate is 1.054 Å. In addition, comparison of SUMO-1 NMR structure and SMT3 crystal structure showed that the r.m.s. deviation of C£\ coordinates is 2.736 Å. Hence, the structures of SUMO-2 and SMT3 are more similar each other than those of SUMO-1and SMT3.