Suggestive evidence for the involvement of the second calcium and surface loop in interfacial binding: monoclinic and trigonal crystal structures of a quadruple mutant of phospholipase A2.

The crystal structures of the monoclinic and trigonal forms of the quadruple mutant K53,56,120,121M of recombinant bovine pancreatic phospholipase A2 (PLA2) have been solved and refined at 1.9 and 1.1 A resolution, respectively. Interestingly, the monoclinic form reveals the presence of the second calcium ion. Furthermore, the surface-loop residues are ordered and the conformation of residues 62-66 is similar to that observed in other structures containing the second calcium ion. On the other hand, in the trigonal form the surface loop is disordered and the second calcium is absent. Docking studies suggest that the second calcium and residues Lys62 and Asp66 from the surface loop could be involved in the interaction with the polar head group of the membrane phospholipid. It is hypothesized that the two structures of the quadruple mutant, monoclinic and trigonal, represent the conformations of PLA2 at the lipid interface and in solution, respectively. A docked structure with a phospholipid molecule and with a transition-state analogue bound, one at the active site coordinating to the catalytic calcium and the other at the second calcium site, but both at the i-face, is presented.

Atomic resolution structure of the double mutant (K53,56M) of bovine pancreatic phospholipase A2.

The structure of the double mutant K53,56M has previously been refined at 1.9 A resolution using room-temperature data. The present paper reports the crystal structure of the same mutant K53,56M refined against 1.1 A data collected using synchrotron radiation. A total of 116 main-chain atoms from 29 residues and 44 side chains are modelled in alternate conformations. Most of the interfacial binding residues are found to be disordered and alternate conformations could be recognized. The second calcium ion-binding site residue Glu92 adopts two alternate conformations. The minor and major conformations of Glu92 correspond to the second calcium ion bound and unbound states.

Third calcium ion found in an inhibitor-bound phospholipase A2.

The lipolytic enzyme phospholipase A2 plays a crucial role in lipid metabolism and catalyzes hydrolysis of the fatty-acid ester bond at the sn-2 position of phospholipids. Here, the crystal structure (1.7 A resolution) of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 complexed with an organic molecule, p-methoxybenzoic acid (anisic acid), is reported. Residues 60-70 (the surface-loop residues) are ordered and adopt conformations which are different from those normally found in structures in which a second calcium ion is present. It is interesting to note that for the first time a third calcium ion has been identified. In addition, four Tris (2-amino-2-hydroxymethyl-1,3-propanediol) molecules were located. It is believed that one of the Tris molecules plays a role in clamping the third calcium ion and that another is involved in controlling the dynamics of the surface loop through hydrogen bonds.

Atomic resolution (0.97 A) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2.

The enzyme phospholipase A2 catalyzes the hydrolysis of the sn-2 acyl chain of phospholipids, forming fatty acids and lysophospholipids. The crystal structure of a triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 in which the lysine residues at positions 53, 56 and 121 are replaced recombinantly by methionines has been determined at atomic resolution (0.97 A). The crystal is monoclinic (space group P2), with unit-cell parameters a = 36.934, b = 23.863, c = 65.931 A, beta = 101.47 degrees. The structure was solved by molecular replacement and has been refined to a final R factor of 10.6% (Rfree = 13.4%) using 63,926 unique reflections. The final protein model consists of 123 amino-acid residues, two calcium ions, one chloride ion, 243 water molecules and six 2-methyl-2,4-pentanediol molecules. The surface-loop residues 60-70 are ordered and have clear electron density.

Somatic INK4a-ARF locus mutations: a significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck.

The INK4a-ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor-suppressor genes. The p16(INK4a) (p16) tumor-suppressor gene product is a negative regulator of cyclin-dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14(ARF) tumor-suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G(1) arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0-10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14(ARF) genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single-stranded conformational polymorphism (SSCP) analysis termed "cold" SSCP. Exons 1alpha, 1beta, and 2 of INK4a/ARF were amplified using intron-based primers or a combination of intron- and exon-based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1alpha. No mutations were found in exon 1beta. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14(ARF) proteins. Specific somatic alterations included microdeletions or insertions (nine of 22, 41%), a microrearrangement (one of 22, 5%), and single nucleotide substitutions (12 of 22, 56%). In addition, we analyzed the functional characteristics of seven unique mutant p16 proteins identified in this study by assessing their ability to inhibit cyclin-dependent kinase 4 activity. Six of the seven mutant proteins tested exhibited reduced function compared with wild-type p16, ranging from minor decreases of function (twofold to eightfold) in four samples to total loss of function (29- to 38-fold decrease) in two other samples. Overall, somatic mutation of the INK4a/ARF tumor suppressor locus, resulting in functionally deficient p16 and possibly p14(ARF) proteins, seems to be a prevalent event in the development of SCCHN. Mol. Carcinog. 30:26-36, 2001.

Structural basis of the anionic interface preference and kcat* activation of pancreatic phospholipase A2.

Pancreatic phospholipase A(2) (PLA2) shows a strong preference for the binding to the anionic interface and a consequent allosteric activation. In this paper, we show that virtually all the preference is mediated through 3 (Lys-53, -56, and -120) of the 12 cationic residues of bovine pancreatic PLA2. The lysine-to-methionine substitution enhances the binding of the enzyme to the zwitterionic interface, and for the K53,56,120M triple mutant at the zwitterionic interface is comparable to that for the wild type (WT) at the anionic interface. In the isomorphous crystal structure, the backbone folding of K53,56M K120,121A and WT are virtually identical, yet a significant change in the side chains of certain residues, away from the site of substitution, mostly at the putative contact site with the interface (i-face), is discernible. Such reciprocity, also supported by the spectroscopic results for the free and bound forms of the enzyme, is expected because a distal structural change that perturbs the interfacial binding could also affect the i-face. The results show that lysine-to-methionine substitution induces a structural change that promotes the binding of PLA2 to the interface as well as the substrate binding to the enzyme at the interface. The kinetic results are consistent with a model in which the interfacial Michaelis complex exists in two forms, and the complex that undergoes the chemical step is formed by the charge compensation of Lys-53 and -56. Analysis of the incremental changes in the kinetic parameters shows that the charge compensation of Lys-53 and -56 contributes to the activation and that of Lys-120 contributes only to the structural change that promotes the stability of the Michaelis complex at the interface. The charge compensation effects on these three residues also account for the differences in the anionic interface preference of the evolutionarily divergent secreted PLA2.

Tumor suppressor INK4: quantitative structure-function analyses of p18INK4C as an inhibitor of cyclin-dependent kinase 4.

We report the first detailed structure-function analyses of p18INK4C (p18), which is a homologue of the important tumor suppressor p16INK4A (p16). Twenty-four mutants were designed rationally. The global conformations of the mutants were characterized by NMR, while the function was assayed by inhibition of cyclin-dependent kinase 4 (CDK4). Most of these mutants have unperturbed global structures, thus the changes in their inhibitory abilities can be attributed to the mutated residues. The important results are summarized as follows: (a) some residues at loops 1 and 2, but not 3, are important for the inhibitory function of p18, similar to the results for p16; (b) two residues at the first helix-turn-helix motif and two at the third are important for inhibition; (c) while the results generally agree with the prediction based on the crystal structures of p16-CDK6 and p19-CDK6 binary complexes, there are significant differences in a few residues, suggesting that the interactions in the binary complexes may not accurately represent the interactions in the ternary complexes (in the presence of cyclin D2); (d) most importantly, the extra loop of p18 appears to contribute to the function of p18, even though the crystal structure of the p19INK4D-CDK6 complex indicates no interactions involving this loop; (e) detailed analyses of the crystal structures and the functional results suggest that there are notable differences in the interactions between different members of the INK4 family and CDKs.

Structural analysis of phospholipase A2 from functional perspective. 2. Characterization of a molten globule-like state induced by site-specific mutagenesis.

Previous NMR studies have shown that many phospholipase A2 (PLA2, from bovine pancreas, overexpressed in Escherichia coli) mutants display some properties reminiscent of a molten globule state. Further NMR analyses for some of the mutants indicated that formation of the "molten globule-like state" is a pH-dependent phenomenon. The mutants I9Y and I9F showed perturbed NMR properties throughout the pH range studied, while the mutants H48A and C44A/C105A displayed native-like spectra at neutral pH but molten globule-like ones under acidic conditions, with a "transition pH" around 4. On the other hand, wild-type PLA2 exhibits exceptional pH stability and turns into a similar molten globule-like state only under highly acidic conditions such as 1 M HCl. The H48A mutant was used to rigorously establish the property of the molten globule-like state of PLA2 mutants. The results of far-UV CD, near-UV CD, and ANS-binding fluorescence suggest that H48A retains native-like secondary structures but loses tertiary structure during the conformational transition. However, the tertiary structure is not completely lost, as evidenced by the retention of some long-range NOEs in two-dimensional NOESY spectra. The conclusion was further substantiated by three-dimensional NOESY-HSQC experiments on a 15N-labeled H48A sample. It was revealed that the molten globule-like state at mildly acidic pH retained some rigid tertiary structure, which consisted of partial alpha-helix II (Y52-L58), alpha-helix III (D59-V63), beta-wing (S74-S85) and partial alpha-helix IV (A90-N97). These residual tertiary structures grouped in half of the protein could be attributed to stabilization by some of the disulfide bonds. The extreme sensitivity of the PLA2 structure to site-directed mutagenesis is unprecedented. It is interesting to note that most of the functional residues (the active site, the hydrophobic channel, the interfacial binding site, and the calcium-binding loop) are located in the remainder of the protein, which is well disrupted in tertiary interactions.

Tumor suppressor INK4: determination of the solution structure of p18INK4C and demonstration of the functional significance of loops in p18INK4C and p16INK4A.

Since the structures of several ankyrin-repeat proteins including the INK4 (inhibitor of cyclin-dependent kinase 4) family have been reported recently, the detailed structures and the functional roles of the loops have drawn considerable interest. This paper addresses the potential importance of the loops of ankyrin-repeat proteins in three aspects. First, the solution structure of p18INK4C was determined by NMR, and the loop structures were analyzed in detail. The loops adapt nascent antiparallel beta-sheet structures, but the positions are slightly different from those in the crystal structure. A detailed comparison between the solution structures of p16 and p18 has also been presented. The determination of the p18 solution structure made such detailed comparisons possible for the first time. Second, the [1H,15N]HSQC NMR experiment was used to probe the interactions between p18INK4C and other proteins. The results suggest that p18INK4C interacts very weakly with dna K and glutathione S-transferase via the loops. The third aspect employed site-specific mutagenesis and functional assays. Three mutants of p18 and 11 mutants of p16 were constructed to test functional importance of loops and helices. The results suggest that loop 2 is likely to be part of the recognition surface of p18INK4C or p16INK4A for CDK4, and they provide quantitative functional contributions of specific residues. Overall, our results enhance understanding of the structural and functional roles of the loops in INK4 tumor suppressors in particular and in ankyrin-repeat proteins in general.