Protein crystallization phosphorylation

Molecular insight into the protein conformation states of Src kinase has been revealed in a series of x-ray crystal structures of the Src SH3-SH2-kinase domain that depict Src in its inactive conformation [7]. This form maintains a closed structure, in which the tyrosine-phosphorylated (Tyr527) C-terminal tail is bound to the SH2 domain (Fig. 2). The x-ray data also reveal binding of the SH3 domain to the SH2-kinase linker [adopts a polyproline type II (PP II) helical conformation], providing additional intramolecular interactions to stabilize the inactive conformation. Collectively, these interactions cause structural changes within the catalytic domain of the protein to compromise access of substrates to the catalytic site and its associated activity. Significantly, these x-ray structures provided the first direct evidence that the SH2 domain plays a key role in the self-regulation of Src. 

The study was performed on a model system based on the crystal structure of Cdc42-Cdc42GAP complexed with GDP and A1F3 [60], which can be considered a TS mimic of phosphoryl transfer [61, 62], A large model system (Fig. 2.6) was required to properly take into account the effect on the reagents of the electrostatic field of the protein. It comprised all the amino acids directly interacting with the triphosphate moiety, the Mg2+ cation with its own coordination shell, and A1F3 replaced by the PO3 moiety. 

The inhibition of SuSy by the divalent cations (fCi 15 aM), Zn (fC 25 aM), and Ni (fti 37 aM) was exploited for further purification of SuSyl by immobilized metal affinity chromatography (IMAC). A subsequent gel filtration yielded homogeneous SuSyl suitable for crystallization experiments [24]. The protein chemical characterization revealed a homotetrameric organization of the 93 kDa subunit. Our kinetic data for the cleavage reaction and preliminary immunoblot analysis for phosphoserine suggested that SuSyl may be phosphorylated in the yeast expression system [28]. 

In subsequent years, much evidence has been adduced to support this mechanism. Alkaline phosphatase and, by analogy, other serine enzymes, are directly phosphorylated on serine serine phosphate is not an artifact (Kennedy and Koshland, 1957). In the presence of nitrophenyl acetate, chymotrypsin is acetylated on serine, and the resulting acetylchymotrypsin has been isolated (Balls and Aldrich, 1955 Balls and Wood, 1956). Similarly, the action of p-nitrophenyl pivalate gave rise to pivaloyl chymotrypsin, which could be crystallized (Balls et al., 1957). Neurath and workers showed that acetylchymotrypsin is hydrolyzed at pH 5.5, but that it is reversibly denatured by 8 M urea the denatured derivative is inert to hydrolysis and even to hydroxylamine, whereas the renatured protein, obtained by... 

In the analysis of the structural data of other protein kinases, it is noted that only cAPK has been crystallized with its specific peptide inhibitor. Nevertheless, three other structures of protein kinases compared with the structure of the cAPK-PKI complex provide substantial evidence for the conservation of the substrate binding cleft. The substrate binding cleft of the phosphorylase kinase structure has been analyzed in detail and it is clear that all amino acids of the known specific substrate can be built into the PKI model and all required corresponding charges can be found in the cleft of the phosphorylase kinase structure. In the CK-1 structure determined without a peptide, the requirement of the peptide specificity resides on the P-3 site, which has to be phosphorylated. An analysis of the surface charges of the cleft of the CK-1 structure reveals the exact correspondence of the residues required to interact with a phosphorylated substrate at this site. 

Adipocyte lipid-binding protein (ALBP) is the adipocyte member of an intracellular hydrophobic ligand-binding protein family. ALBP is phosphorylated by the insulin receptor kinase upon insulin stimulation. The crystal structure of recombinant murine ALBP has been determined and refined to 2.5 A. The final R-factorfor the model is 0.18 with good canonical properties. 

The system may be regarded as involving a Na+/Mg2+ co-catalysed phosphorylation step and a K+ catalysed dephosphorylation. Each phosphorylation/dephosphorylation step involves a pseudorotation of an Mg2+-stabilised 5-coordinate intermediate, resulting in transport of the alkali metal cations. The cation transport ability of the enzyme is a direct result of the enzymatic reactivity of the protein. There are three binding sites with high Na+ affinity and two with K+ affinity (occupied by Rb+ in the crystal structure determination). The structure (which is of the E2K state of the system) reveals that carboxy end of the a-subunit is held in a pocket in between transmembrane helices and acts as an unusual regulating element that controls sodium affinity and may be influenced by the membrane potential. 

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