Co-crystal structures of PKG Iß (92-227) with cGMP and cAMP reveal the molecular details of cyclic-nucleotide binding.

BACKGROUND: Cyclic GMP-dependent protein kinases (PKGs) are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate most of the effects of cGMP elevating drugs, such as nitric oxide-releasing agents and phosphodiesterase inhibitors which are used for the treatment of angina pectoris and erectile dysfunction, respectively. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the mechanism of cyclic nucleotide binding to PKG by determining crystal structures of the amino-terminal cyclic nucleotide-binding domain (CNBD-A) of human PKG I bound to either cGMP or cAMP. We also determined the structure of CNBD-A in the absence of bound nucleotide. The crystal structures of CNBD-A with bound cAMP or cGMP reveal that cAMP binds in either syn or anti configurations whereas cGMP binds only in a syn configuration, with a conserved threonine residue anchoring both cyclic phosphate and guanine moieties. The structure of CNBD-A in the absence of bound cyclic nucleotide was similar to that of the cyclic nucleotide bound structures. Surprisingly, isothermal titration calorimetry experiments demonstrated that CNBD-A binds both cGMP and cAMP with a relatively high affinity, showing an approximately two-fold preference for cGMP. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that CNBD-A binds cGMP in the syn conformation through its interaction with Thr193 and an unusual cis-peptide forming residues Leu172 and Cys173. Although these studies provide the first structural insights into cyclic nucleotide binding to PKG, our ITC results show only a two-fold preference for cGMP, indicating that other domains are required for the previously reported cyclic nucleotide selectivity.

The presence of monoglucosylated N196-glycan is important for the structural stability of storage protein, arylphorin.

Although N-glycosylation has been known to increase the stability of glycoproteins, it is difficult to assess the structural importance of glycans in the stabilization of glycoproteins. APA (Antheraea pernyi arylphorin) is an insect hexamerin that has two N-glycosylations at Asn196 and Asn344 respectively. The glycosylation of Asn344 is critical for the folding process; however, glycosylation of Asn196 is not. Interestingly, the N196-glycan (glycosylation of Asn196) remains in an immature form (Glc1Man9GlcNAc2). The mutation of Asn196 to glutamine does not change the ecdysone-binding activity relative to that of the wild-type. In the present study, we determined the crystal structure of APA, and all sugar moieties of the N196-glycan were clearly observed in the electron-density map. Although the sugar moieties of the glycan generally have high structural flexibility, most sugar moieties of the N196-glycan were well organized in the deep cleft of the subunit interface and mediated many inter- and intrasubunit hydrogen bonds. Analytical ultracentrifugation and GdmCl (guanidinium chloride) unfolding experiments revealed that the presence of the N196-glycan was important for stabilizing the hexameric state and overall stability of APA respectively. Our results could provide a structural basis for studying not only other glycoproteins that carry an immature N-glycan, but also the structural role of N-glycans that are located in the deep cleft of a protein.

Crystal structures of substrate and inhibitor complexes of ribose 5-phosphate isomerase A from Vibrio vulnificus YJ016.

Ribose-5-phosphate isomerase A (RpiA) plays an important role in interconverting between ribose-5-phosphate (R5P) and ribulose-5-phosphate in the pentose phosphate pathway and the Calvin cycle. We have determined the crystal structures of the open form RpiA from Vibrio vulnificus YJ106 (VvRpiA) in complex with the R5P and the closed form with arabinose-5-phosphate (A5P) in parallel with the apo VvRpiA at 2.0 A resolution. VvRpiA is highly similar to Eschericihia coliRpiA, and the VvRpiA-R5P complex strongly resembles the E. coli RpiA-A5P complex. Interestingly, unlike the E. coli RpiA-A5P complex, the position of A5P in the VvRpiA-A5P complex reveals a different position than the R5P binding mode. VvRpiA-A5P has a sugar ring inside the binding pocket and a phosphate group outside the binding pocket: By contrast, the sugar ring of A5P interacts with the Asp4, Lys7, Ser30, Asp118, and Lys121 residues; the phosphate group of A5P interacts with two water molecules, W51 and W82.