Co-crystallization of the human nuclear cap-binding complex with a m7GpppG cap analogue using protein engineering.
The nuclear cap-binding complex (CBC) binds the 7-methyl-G(5')ppp(5')N cap structure at the 5' end of pre-messenger and uracil-rich small nuclear RNAs in the nucleus. It mediates interaction of these capped RNAs with various nuclear machineries involved in RNA maturation and is co-exported with them to the cytoplasm. The structure of human CBC, which comprises the subunits CBP20 and CBP80, has previously been determined in a mildly trypsinated form which can no longer bind the cap. Here, the engineering and crystallization of two variant CBCs with deletions in CBP80 which do not affect function are described. A complex with a small N-terminal deletion in CBP80 was crystallized in space group C2 with one complex per asymmetric unit. The crystals diffract to 2 A resolution and give the first structure of intact but cap-free CBC. An additional internal deletion in CBP80 of a prominent solvent-exposed coiled coil gives rise to a more compact complex. This was co-crystallized with the cap analogue m(7)GpppG in two different crystal forms which could grow in the same drop. Form 1 belongs to space group P3(1)21 with one complex per asymmetric unit and diffracts to 2.15 A resolution. Form 2 belongs to space group P2(1)2(1)2(1) with two complexes per asymmetric unit and diffracts to 2.3 A resolution. In both forms, strong extra electron density is observed for the cap analogue and for the N- and C-terminal extensions of CBP20 which was absent or disordered in all previous structures.
Mazza
Catherine
C
European Molecular Biology Laboratory, Grenoble Outstation, c/o ILL, BP 181, F-38042 Grenoble CEDEX 9, France.
Segref
Alexandra
A
Mattaj
Iain W
IW
Cusack
Stephen
S
D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D017931:DNA Primers; D015226:Dinucleoside Phosphates / Q000737:chemistry*; D006801:Humans; D039502:Nuclear Cap-Binding Protein Complex; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015202:Protein Engineering; D012315:RNA Caps / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins
C100383:CBC2 protein, S cerevisiae; D017931:DNA Primers; D015226:Dinucleoside Phosphates; D039502:Nuclear Cap-Binding Protein Complex; D010750:Phosphoproteins; D012315:RNA Caps; D029701:Saccharomyces cerevisiae Proteins; C062229:7-methyl-diguanosine triphosphate