Dissection of protein-protein interaction and CDK4 inhibition in the oncogenic versus tumor suppressing functions of gankyrin and P16

Protein-protein interactions usually involve a large number of residues; thus it is difficult to elucidate functional and structural roles of specific residues located in the interface. This problem is particularly challenging for ankyrin repeat proteins (ARs), which consist of linear arrays of small repeating units and play critical roles in almost every life process via protein-protein interactions, because the residues involved are discontinuously dispersed in both the ARs and their partners. Our previous studies showed that while both specific CDK4 inhibitor p16INK4A (P16) and gankyrin bind to cyclin-dependent kinase 4 (CDK4) in similar fashion, only P16 inhibits the kinase activity of CDK4. While this could explain why P16 is a tumor suppressor and gankyrin is oncogenic, the structural basis of these contrasting properties was unknown. Here we show that a double mutant of gankyrin, L62H/I79D, inhibits the kinase activity of CDK4, similar to P16, and such CDK4-inhibtory activity is associated with the I79D but not L62H mutation. In addition, mutations at I79 and L62 bring about a moderate decrease in the stability of gankyrin. Further structural and biophysical analyses suggest that the substitution of Ile79 with Asp leads to local conformational changes in loops I-III of gankyrin. Taken together, our results allow the dissection of the "protein-protein binding" and "CDK4 inhibition" functions of P16, show that the difference between tumor suppressing and oncogenic functions of P16 and gankyrin, respectively, mainly resides in a single residue, and provide structural insight to the contrasting biological functions of the two AR proteins.     

An NF-kappaB-specific inhibitor, IkappaBalpha, binds to and inhibits cyclin-dependent kinase 4

IkappaBalpha, a protein composed of six ankyrin repeats, is a specific inhibitor of nuclear factor kappaB (NF-kappaB) and functions in signal transductions in many different cell types. Using both in vivo yeast two-hybrid assays and in vitro activity and binding assays, we showed that IkappaBalpha binds to cyclin-dependent kinase 4 (CDK4) specifically and inhibits its kinase activity. The potencies of binding and inhibition of IkappaBalpha are comparable to those of INK4 proteins, the specific CDK4 inhibitors that also contain ankyrin repeats. Furthermore, we showed that INK4 proteins and IkappaBalpha compete with each other for binding to CDK4. These results led us to propose a hypothesis that there is cross talk between the NF-kappaB/IkappaBalpha pathway and the p16/CDK4/Rb pathway in cells, and that IkappaBalpha could substitute for the CDK4-inhibiting function of p16, a tumor suppressor frequently inactivated in human tumors. To further understand the structural basis of IkappaBalpha-CDK binding, we used different mutants of CDK4 to show that there are notable differences between IkappaBalpha and INK4 proteins in CDK4 binding since the binding is affected differently by different CDK4 mutations. We also demonstrated that the interaction of IkappaBalpha with CDK4 is different from that with its NF-kappaB. While most of the contacts contributing to NF-kappaB binding are located within the last two C-terminal ankyrin repeats and the loop region bridging them, the first four ankyrin repeats at the N-terminus are responsible for CDK4 binding and inhibition.     

Solution structure of the human oncogenic protein gankyrin containing seven ankyrin repeats and analysis of its structure--function relationship

Human gankyrin (226 residues, 24.4 kDa) is a liver oncoprotein that plays an important role in the development of human hepatocellular carcinomas. In this paper, its solution structure is reported, which is the largest ankyrin protein ever determined by NMR. The highly degenerate primary sequences of the seven ankyrin repeats presented a major challenge, which was overcome by combined use of TROSY experiments, perdeuterated samples, isotope-filtered NMR experiments, and residual dipolar couplings. The final structure was of high quality, with atomic rmsds for the backbone (N, C', and C(alpha)) and all heavy atoms (residues 4-224) of 0.69 +/- 0.09 and 1.04 +/- 0.09 A, respectively. Detailed analyses of NMR data suggested that the conserved TPLH motifs play important structural roles in stabilizing the repeating ankyrin scaffold. Gankyrin is conformationally more stable than the tumor suppressor p16(INK4A), possibly due to the structural roles of conserved residues evidenced by slowly exchanging backbone amides as well as hydrogen bonding networks involving labile side chain protons. Structural comparison with p16(INK4A) identified several residues of gankyrin that are potentially important for CDK4 binding, whereas observation of the thiol proton of C180 indicated a well-structured Rb-binding site in the helical region of the sixth ankyrin repeat. Interestingly, the CDK4-binding site and Rb-binding site located in N- and C-terminal regions, respectively, are separated by comparatively more stable ankyrin repeats and highly condensed positive surface charge. These results and analyses will shed light on the structural basis of the function of human gankyrin.     

Novel insights into the INK4-CDK4/6-Rb pathway: counter action of gankyrin against INK4 proteins regulates the CDK4-mediated phosphorylation of Rb

The newly discovered oncogenic protein gankyrin, which contains six ankyrin repeats, has been reported to be involved in the phosphorylation and degradation of the retinoblastoma gene product, Rb. Using in vitro systems, we have identified a peptide fragment of gankyrin, 176LHLACDEERN185, which is responsible for binding of gankyrin to Rb. We further demonstrated a different mechanism for gankyrin to facilitate the phosphorylation of Rb, by binding with cyclin-dependent kinase 4 (CDK4). This binding does not inhibit the Rb-phosphorylating kinase activity of CDK4, but it competes with p16 binding to CDK4 and counteracts the inhibitory function of p16. We then showed that binding of gankyrin to CDK4 and the consequent counter action of p16 function were not affected by the Rb-binding peptide 176LHLACDEERN185, indicating that the two mechanisms are independent. They also involve different structural regions of gankyrin. While the Rb-binding motif is located at the fifth ankyrin repeat, truncation mutants of gankyrin, with the first three or four ankyrin repeats remaining, are sufficient for binding to CDK4 and for counteracting the inhibitory function of p16. These results demonstrate the potential importance of gankyrin in cell cycle control and tumorigenesis and suggest an expanded INK4-CDK4/6-Rb pathway.     

Arginine methylation inhibits the binding of proline-rich ligands to Src homology 3, but not WW, domains

Src homology 3 (SH3) and WW domains are known to associate with proline-rich motifs within their respective ligands. Here we demonstrate that the proposed adapter protein for Src kinases, Sam68, is a ligand whose proline-rich motifs interact with the SH3 domains of p59(fyn) and phospholipase Cgamma-1 as well as with the WW domains of FBP30 and FBP21. These proline-rich motifs, in turn, are flanked by RG repeats that represent targets for the type I protein arginine N-methyltransferase. The asymmetrical dimethylation of arginine residues within these RG repeats dramatically reduces the binding of the SH3 domains of p59(fyn) and phospholipase Cgamma-1, but has no effect on their binding to the WW domain of FBP30. These results suggest that protein arginine methylation can selectively modulate certain protein-protein interactions and that mechanisms exist for the irreversible regulation of SH3 domain-mediated interactions.     

Comparative structural analysis of the binding domain of follicle stimulating hormone receptor

Proteins with leucine-rich repeats (LRRs) specialize in mediating protein-protein interactions. The hormone binding portion of the receptor for follicle stimulating hormone (FSH) is an LRR protein by sequence, and the crystal structure of this domain from human FSH receptor in a complex with FSH shows that it does indeed have an LRR structure. It differs from other LRR domains, however, in being an all-beta protein composed of highly irregular repeats and having only slight overall curvature. Despite these distinctions and a superficial resemblance to beta-helical proteins, the binding domain of FSH receptor clearly is an LRR protein. The structure does consist of two parts with distinctively different curvatures. Comparison with the structures of other LRR-containing proteins shows a correlation between curvature and main-chain hydrogen bonding pattern of the parallel beta-sheet. The hormone-binding site is located at the concave surface of the receptor structure, a feature common to proteins with LRR motifs. Analysis of the ligand-binding site of LRR-containing proteins reveals that they generally utilize extensive interface area and a large number of charged residues to facilitate high-affinity protein-protein interactions.     

The leucine-rich repeat as a protein recognition motif

Leucine-rich repeats (LRRs) are 20-29-residue sequence motifs present in a number of proteins with diverse functions. The primary function of these motifs appears to be to provide a versatile structural framework for the formation of protein-protein interactions. The past two years have seen an explosion of new structural information on proteins with LRRs. The new structures represent different LRR subfamilies and proteins with diverse functions, including GTPase-activating protein rna1p from the ribonuclease-inhibitor-like subfamily; spliceosomal protein U2A', Rab geranylgeranyltransferase, internalin B, dynein light chain 1 and nuclear export protein TAP from the SDS22-like subfamily; Skp2 from the cysteine-containing subfamily; and YopM from the bacterial subfamily. The new structural information has increased our understanding of the structural determinants of LRR proteins and our ability to model such proteins with unknown structures, and has shed new light on how these proteins participate in protein-protein interactions.     

The 1.1-angstrom structure of the spindle checkpoint protein Bub3p reveals functional regions

Bub3p is a protein that mediates the spindle checkpoint, a signaling pathway that ensures correct chromosome segregation in organisms ranging from yeast to mammals. It is known to function by co-localizing at least two other proteins, Mad3p and the protein kinase Bub1p, to the kinetochore of chromosomes that are not properly attached to mitotic spindles, ultimately resulting in cell cycle arrest. Prior sequence analysis suggested that Bub3p was composed of three or four WD repeats (also known as WD40 and beta-transducin repeats), short sequence motifs appearing in clusters of 4-16 found in many hundreds of eukaryotic proteins that fold into four-stranded blade-like sheets. We have determined the crystal structure of Bub3p from Saccharomyces cerevisiae at 1.1 angstrom and a crystallographic R-factor of 15.3%, revealing seven authentic repeats. In light of this, it appears that many of these repeats therefore remain hidden in sequences of other proteins. Analysis of random and site-directed mutants identifies the surface of Bub3p involved in checkpoint function through binding of Bub1p and Mad3p. Sequence alignments indicate that these surfaces are mostly conserved across Bub3 proteins from diverse species. A structural comparison with other proteins containing WD repeats suggests that these folds may bind partner proteins using similar surface areas on the top and sides of the propeller. The sequences composing these regions are the most divergent within the repeat across all WD repeat proteins and could potentially be modulated to provide specificity in partner protein binding without perturbation of the core structure.     

Insights into signal transduction revealed by the low resolution structure of the FixJ response regulator

P19(INK4d) is a tumor suppressing protein and belongs to a family of cyclin D-dependent kinase inhibitors of CDK4 and CDK6, which play a key role in human cell cycle control. P19 comprises ten alpha-helices arranged sequentially in five ankyrin repeats forming an elongated structure. This rather simple topology, combined with its physiological function, makes p19 an interesting model protein for folding studies. Urea-induced unfolding transitions monitored by far-UV CD and phenylalanine fluorescence coincide and suggest a two-state mechanism for equilibrium unfolding. Unfolding of p19 followed by 2D (1)H-(15)N HSQC spectra revealed a third species at moderate urea concentrations with a maximum population of about 30 % near 3.2 M urea. It shows poor chemical shift dispersion, but cross-peaks emerge for some residues that are distinct from the native or unfolded state. This equilibrium intermediate either arises only at high protein concentrations (as in the NMR experiment) or has similar optical properties to the unfolded state. Stopped-flow far-UV CD experiments at various urea concentrations revealed that alpha-helical structure is formed in three phases, of which only the fastest phase (10 s(-1)) depends upon the urea concentration. The kinetic of the slowest phase (0.017 s(-1)) can be resolved by 1D real-time NMR and accelerated by cyclophilin. It is limited in rate by prolyl isomerization, and native-like ordered structure cannot form prior to this isomerization. The two fast phases lead to 83 % native protein within the dead time of the NMR experiment. In contrast to p16(INK4a), which exhibits only a marginal stability and high unfolding rates, p19 shows the expected stability for a protein of this size with a clear kinetic barrier between the unfolded and folded state. Therefore, p19 might complement the function of less stable INK4 inhibitors in cell cycle control under unfavorable conditions.     

Crystal structure of the homolog of the oncoprotein gankyrin, an interactor of Rb and CDK4/6

The oncoprotein gankyrin plays a central role in tumorigenesis and cell proliferation. Gankyrin interacts with the retinoblastoma tumor suppressor (Rb) and cyclin-dependent kinase 4/6 (CDK4/6), increases phosphorylation at specific residues of Rb by CDK4/6 in vivo, and promotes tumorigenesis. The phosphorylation of Rb by CDK4/6 leads to the deregulation of the cell cycle during G1/S transition. Although how phosphorylation occurs on Rb has been studied extensively, the mechanism of site-specific phosphorylation of Rb remains unclear due to a lack of information on the structural arrangement of Rb and CDK4/6. Here, we have determined and refined to 2.3-A resolution the crystal structure of a gankyrin homolog, the non-ATPase subunit 6 (Nas6p) of the proteasome from yeast. The crystal structure reveals that Nas6p contains seven ankyrin repeats. The number of the repeats is different from that predicted from the primary structure. Nas6p also possesses an unusual curved structure with two acidic regions at the N- and C-terminal regions separated by one basic region, suggesting that it has at least two functional surfaces. The tertiary structure of Nas6p, together with the previous biochemical studies, indicates that the CDK4/6 and Rb binding surfaces of gankyrin are located at the N- and C-terminal regions, respectively, and face the same side of gankyrin. These observations suggest that gankyrin brings Rb and CDK4/6 together through gankyrin-Rb and gankyrin-CDK4/6 interactions and determines the relative positioning of the substrate (Rb) and the enzyme (CDK4/6). Our findings provide mechanistic insight into site-specific phosphorylation of Rb caused by CDK4/6.     

Unfolding transition state and intermediates of the tumor suppressor p16INK4a investigated by molecular dynamics simulations

The ankyrin repeat is one of the most common protein motifs and is involved in protein-protein interactions. It consists of 33 residues that assume a beta-hairpin helix-loop-helix fold. Mutagenesis and kinetic experiments (Phi-value analysis of the folding transition state) have shown that the tumor suppressor p16(INK4a), a four-repeat protein, unfolds sequentially starting from the two N-terminal repeats. Here, the flexibility of p16(INK4a) at room temperature and its unfolding mechanism at high temperature have been investigated by multiple molecular dynamics runs in explicit water for a total simulation time of 0.65 micros. The transition state ensemble (TSE) of p16(INK4a) was identified by monitoring both the deviation from the experimental Phi values and sudden conformational changes along the unfolding trajectories. Conformations in the TSE have a mainly unstructured second repeat whereas the other repeats are almost completely folded. A rigid-body displacement of the first repeat involving both a rotation and translation is observed in all molecular dynamics simulations at high temperature. The Trp(15), Pro(75), and Ala(76) side-chains are more buried in the TSE than the native state. The sequential unfolding starting at the second repeat is in agreement with the mutagenesis studies whereas the displacement of the first repeat and the presence of nonnative interactions at the TSE are simulation results which supplement the experimental data. Furthermore, the unfolding trajectories reveal the presence of two on-pathway intermediates with partial alpha-helical structure. Finally, on the basis of the available experimental and simulation results we suggest that in modular proteins the shift of the folding TSE toward the native structure upon reduction of the number of tandem repeats is consistent with the Hammond effect.     

An in silico approach for the discovery of CDK5/p25 interaction inhibitors

The lack of selectivity of all existing ATP competitive inhibitors for a single cyclin-dependent kinase (CDK) has led us to redirect the structure-based molecule design from targeting the classic ATP-binding pocket in CDK5 toward the CDK5/p25 interface. The aim was to seek novel inhibition mechanisms to interrupt protein-protein interactions. A combined strategy of alanine-scanning calculations for locating binding sites, virtual screening for small molecules, molecular dynamics simulations for examining the binding stability of virtual screening hits and bio-assays for testing the level of inhibition was set up and used to explore novel inhibitors capable of interrupting the interactions between the proteins, and consequently of inhibiting the kinase activity. Two compounds were shown to inhibit the complex formation between CDK5 and p25 through p25 binding. They could open avenues for the discovery of new types of structures that prevent interactions between CDK5 and p25 or other CDK and activator proteins, and, more importantly, provide leads in the development of selective inhibitors among CDKs.     

Identification of substrate binding site of cyclin-dependent kinase 5

Cyclin-dependent kinase 5 (CDK5), unlike other CDKs, is active only in neuronal cells where its neuron-specific activator p35 is present. However, it phosphorylates serines/threonines in S/TPXK/R-type motifs like other CDKs. The tail portion of neurofilament-H contains more than 50 KSP repeats, and CDK5 has been shown to phosphorylate S/T specifically only in KS/TPXK motifs, indicating highly specific interactions in substrate recognition. CDKs have been shown to have a high preference for a basic residue (lysine or arginine) as the n+3 residue, n being the location in the primary sequence of a phosphoacceptor serine or threonine. Because of the lack of a crystal structure of a CDK-substrate complex, the structural basis for this specific interaction is unknown. We have used site-directed mutagenesis ("charged to alanine") and molecular modeling techniques to probe the recognition interactions for substrate peptide (PKTPKKAKKL) derived from histone H1 docked in the active site of CDK5. The experimental data and computer simulations suggest that Asp86 and Asp91 are key residues that interact with the lysines at positions n+2 and/or n+3 of the substrates.     

Geometric Similarities of Protein-Protein Interfaces at Atomic Resolution Are Only Observed within Homologous Families: An Exhaustive Structural Classification Study

To elucidate the structural basis of the diversity and universality in protein-protein interactions, an exhaustive all-against-all structural comparison of all known protein interfaces in the Protein Data Bank was performed at atomic resolution. After similar interfaces were clustered, approximately 20,000 structural motifs with at least two members were identified, out of which 3678 motifs consisted of at least 10 interfaces. Except for some trivial interfaces involving single α helices, almost all motifs were found to be confined within single protein families. Furthermore, the interaction partners of each motif were found to be very limited, and, accordingly, the interaction networks of the motifs tend to be small and are much more restricted than the binding sites for small ligand molecules. These findings suggest that, at the level of atomic structures, protein-protein interactions are precisely designed; hence, protein interfaces with multiple interacting partners should involve incompletely overlapping multiple interfaces and/or accommodate structural changes upon binding to their targets.     

Predicting protein-protein interactions on a proteome scale by matching evolutionary and structural similarities at interfaces using PRISM

Prediction of protein-protein interactions at the structural level on the proteome scale is important because it allows prediction of protein function, helps drug discovery and takes steps toward genome-wide structural systems biology. We provide a protocol (termed PRISM, protein interactions by structural matching) for large-scale prediction of protein-protein interactions and assembly of protein complex structures. The method consists of two components: rigid-body structural comparisons of target proteins to known template protein-protein interfaces and flexible refinement using a docking energy function. The PRISM rationale follows our observation that globally different protein structures can interact via similar architectural motifs. PRISM predicts binding residues by using structural similarity and evolutionary conservation of putative binding residue 'hot spots'. Ultimately, PRISM could help to construct cellular pathways and functional, proteome-scale annotation. PRISM is implemented in Python and runs in a UNIX environment. The program accepts Protein Data Bank-formatted protein structures and is available at http://prism.ccbb.ku.edu.tr/prism_protocol/.     

Ankyrin repeats in context with human population variation

Ankyrin protein repeats bind to a wide range of substrates and are one of the most common protein motifs in nature. Here, we collate a high-quality alignment of 7,407 ankyrin repeats and examine for the first time, the distribution of human population variants from large-scale sequencing of healthy individuals across this family. Population variants are not randomly distributed across the genome but are constrained by gene essentiality and function. Accordingly, we interpret the population variants in context with evolutionary constraint and structural features including secondary structure, accessibility and protein-protein interactions across 383 three-dimensional structures of ankyrin repeats. We find five positions that are highly conserved across homologues and also depleted in missense variants within the human population. These positions are significantly enriched in intra-domain contacts and so likely to be key for repeat packing. In contrast, a group of evolutionarily divergent positions are found to be depleted in missense variants in human and significantly enriched in protein-protein interactions. Our analysis also suggests the domain has three, not two surfaces, each with different patterns of enrichment in protein-substrate interactions and missense variants. Our findings will be of interest to those studying or engineering ankyrin-repeat containing proteins as well as those interpreting the significance of disease variants.     

Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity

The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ～35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the analysis of protein-protein interactions for chemical biology and molecular therapeutics.     

Multiple TPR motifs characterize the Fanconi anemia FANCG protein

The genome protection pathway that is defective in patients with Fanconi anemia (FA) is controlled by at least eight genes, including BRCA2. A key step in the pathway involves the monoubiquitylation of FANCD2, which critically depends on a multi-subunit nuclear 'core complex' of at least six FANC proteins (FANCA, -C, -E, -F, -G, and -L). Except for FANCL, which has WD40 repeats and a RING finger domain, no significant domain structure has so far been recognized in any of the core complex proteins. By using a homology search strategy comparing the human FANCG protein sequence with its ortholog sequences in Oryzias latipes (Japanese rice fish) and Danio rerio (zebrafish) we identified at least seven tetratricopeptide repeat motifs (TPRs) covering a major part of this protein. TPRs are degenerate 34-amino acid repeat motifs which function as scaffolds mediating protein-protein interactions, often found in multiprotein complexes. In four out of five TPR motifs tested (TPR1, -2, -5, and -6), targeted missense mutagenesis disrupting the motifs at the critical position 8 of each TPR caused complete or partial loss of FANCG function. Loss of function was evident from failure of the mutant proteins to complement the cellular FA phenotype in FA-G lymphoblasts, which was correlated with loss of binding to FANCA. Although the TPR4 mutant fully complemented the cells, it showed a reduced interaction with FANCA, suggesting that this TPR may also be of functional importance. The recognition of FANCG as a typical TPR protein predicts this protein to play a key role in the assembly and/or stabilization of the nuclear FA protein core complex.