A large number of proteins have shown ability to bind to SUMO (Small Ubiquitin like Modifier) proteins through a short conserved motif called SIM (SUMO Interacting Motif). The work presented here shows that the different SUMO isoforms interact with the hydrophobic core of the SIM by forming an intermolecular b-sheet with the b2 strand of SUMO. This interaction is crucial for SUMO binding, and is modulated by interactions between SUMO and the amino acids flanking the core of the SIM. The SIM can be phosphorylated, providing a possibility for regulating the strength of SUMO binding in the lifetime of a protein. Furthermore, a concentration threshold effect is observed in the binding of the unphosphorylated SIM of PIAS (Protein inhibitor of activated STAT) to SUMO. The dependency on the amino acids flanking the hydrophobic core is stronger in binding to SUMO1 than to SUMO2, providing a mechanism for SUMO isoform discrimination.