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Many proteins contain a peptide 'zip code' that targets them to their destination in the cell. Nuclear zip codes are recognized by one of ten import factors, which transport the signal-containing proteins into the nucleus. One of these transporter proteins, Kapβ2, recognizes the PY nuclear localization signal (NLS), a zip code that is different from the classical short, basic NLS that binds Kapα-Kapβ1.

After solving the structure of Kapβ2 bound to a PY-NLS–containing substrate, Yuh Min Chook and colleagues at the University of Texas Southwestern Medical Center at Dallas defined Kapβ2 signal recognition rules (Lee et al., 2006), and then identified other potential substrates. “We needed to validate these proteins as Kapβ2 substrates, especially in vivo, but this is a cumbersome task,” says Chook. “It would be helpful if we had an inhibitor [of Kapβ2], but there were none.”

Given sufficient manpower and funding, a chemical screen can be a way to find an inhibitor, but this option is not always available to a small, young lab like Chook's. Instead, they found a way to design a peptide inhibitor when they solved the structure of another PY-NLS–containing substrate bound to Kapβ2 (Cansizoglu et al., 2007). A comparison of this complex with the previously solved one revealed that the two signals are structurally different but converge at three spots. These epitopes each bind Kapβ2 quasi-independently and with varying affinities, and most importantly, the binding hot spots in the two NLSs are asymmetrically located.

Armed with this information, Chook and colleagues saw an opportunity to design a tight binder to jam up Kapβ2, and thus inhibit nuclear import via this pathway. Their peptide, M9M, is a chimera of the identified asymmetric hot spots. In HeLa cells, M9M blocks nuclear import of several Kapβ2 substrates because of its tighter binding. “It's so tight that it's not a signal anymore because it can't get released in the nucleus, and so it becomes an inhibitor,” explains Chook.

As well as using M9M to validate potential Kapβ2 substrates, the researchers aim to map the potential redundancy in import networks. In the future, Chook hopes to make a chemical inhibitor but underscores that “it will be very hard to find one small molecule that could block NLS recognition as well as our peptide, but you can presumably identify multiple compounds that bind to the different epitopes and tether them together.”