The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C‐terminally truncated catalytic and two regulatory subunits has been determined at 3.1 Å resolution (Protein Data Bank code: 1JWH). In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C‐terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit as a docking partner for various protein kinases. Furthermore it shows an inter‐domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure.

The heterotetrameric recombinant holoenzyme of human protein kinase CK2 was puri®ed to homogeneity. It degraded spontaneously to a stable and fully active state in which the catalytic subunit was about 5 kDa smaller than the wild type. The degraded enzyme was crystallized using polyethylene glycol 3350 as precipitant. The crystals belong to the hexagonal space group P6 3 . They have unit-cell parameters a = b = 176.0, c = 93.6 A Ê and diffract X-rays to at least 3.5 A Ê resolution. The calculated crystal packing parameter is V M = 3.22 A Ê 3 Da À1 , suggesting that one CK2 tetramer is contained in the asymmetric unit and that the solvent content of the unit cell is 62%.

A search for strategies was conducted in order to obtain a human protein kinase CK2 preparation which would be suitable for crystallization, despite the fact that the recombinant enzyme is abundant and can be readily purified to homogeneity. This seemingly contradiction is based on the fact that the catalytic subunit moiety of the human CK2 holoenzyme is not stable neither as a free subunit nor in the tetrameric complex. All attempts to prevent degradation failed. Hence, alternative approaches were designed in order to avoid this degradation, which was expected to hamper any crystallization efforts severely. One of the approaches chosen was the production of a chimeric holoenzyme made up from a human regulatory subunit and a catalytic subunit from Z. mays. The plant catalytic subunit, in contrast to the human counterpart is very stable and does not undergo this kind of degradation. The second strategy to tackle the problem of instability was to produce the homologous recombinant human CK2 holoenzyme and then, instead of trying to avoid degradation, attempt to accelerate degradation until all catalytic subunit material was converted to the degraded form, i.e. a 40 kDa polypeptide.

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