No abstract

Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. Wildtype neuroglobin has been shown to have six-coordinate heme in both ferric and ferrous states. Substitution of His 96 by alanine leads to complete loss of heme, indicating that His 96 is the proximal ligand. The resonance Raman spectra of M69L and K67T mutants were similar to those of wild-type (WT) neuroglobin in both ferric and ferrous states. By contrast, H64V was six-coordinate high-spin and five-coordinate high-spin in the ferric and ferrous states, respectively, at acidic pH. The spectra were pH-dependent and six-coordinate with the lowspin component dominating at alkaline pH. In a double mutant H64V/K67T, the high-spin component alone was detected in the both ferric and the ferrous states. This implies that His 64 is the endogenous ligand and that Lys 67 is situated nearby in the distal pocket. In the ferrous H64V and H64V/K67T mutants, the (Fe-His) stretching frequency appears at 221 cm ؊1 , which is similar to that of deoxymyoglobin. In the ferrous CO-bound state, the (Fe-CO) stretching frequency was detected at 521 and 494 cm ؊1 in WT, M69L, and K67T, while only the 494 cm ؊1 component was detected in the H64V and H64V/K67T mutants. Thus, the 521 cm ؊1 component is attributed to the presence of polar His 64 . The CO binding kinetics were biphasic for WT, H64V, and K67T and monophasic for H64V/K67T. Thus, His 64 and Lys 67 comprise a unique distal heme pocket in neuroglobin.

Kinesin spindle protein Eg5 is a target for anticancer therapies, and small molecule inhibitors of its ATPase activity have been developed. We herein report for the first time the crystal structure of and biochemical studies on the Eg5 motor domain in complex with a new type of allosteric inhibitor. The biphenyl-type inhibitor PVZB1194 binds to the α4/α6 allosteric pocket 15 Å from the ATP-binding pocket, which differs from conventional allosteric inhibitors that bind to the allosteric L5/α2/α3 pocket of Eg5. Binding of the inhibitor is involved in the neck-linker conformation and also causes conformational changes around the ATP-binding pocket through Tyr104 to affect the interaction of ATP with the pocket. This structure provides useful information for the development of novel types of allosteric drugs as well as a novel insight into the molecular mechanism responsible for regulating the motor activity of kinesins.

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Mitotic arrest deficient 2-like protein 2 (MAD2L2), also termed MAD2B or REV7, is involved in multiple cellular functions including translesion DNA synthesis (TLS), signal transduction, transcription, and mitotic events. MAD2L2 interacts with chromosome alignment-maintaining phosphoprotein (CAMP), a kinetochore-microtubule attachment protein in mitotic cells, presumably through a novel "WK" motif in CAMP. Structures of MAD2L2 in complex with binding regions of the TLS proteins REV3 and REV1 have revealed that MAD2L2 has two faces for protein-protein interactions that are regulated by its C-terminal region; however, the mechanisms underlying the MAD2L2-CAMP interaction and the mitotic role of MAD2L2 remain unknown. Here we have determined the structures of human MAD2L2 in complex with a CAMP fragment in two crystal forms. The overall structure of the MAD2L2-CAMP complex in both crystal forms was essentially similar to that of the MAD2L2-REV3 complex. However, the residue interactions between MAD2L2 and CAMP were strikingly different from those in the MAD2L2-REV3 complex. Furthermore, structure-based interaction analyses revealed an unprecedented mechanism involving CAMP's WK motif. Surprisingly, in one of the crystal forms, the MAD2L2-CAMP complex formed a dimeric structure in which the C-terminal region of MAD2L2 was swapped and adopted an immature structure. The structure provides direct evidence for the dynamic nature of MAD2L2 structure, which in turn may have implications for the protein-protein interaction mechanism and the multiple functions of this protein. This work is the first structural study of MAD2L2 aside from its role in TLS and might pave the way to clarify MAD2L2's function in mitosis.