Structure of the catalytic domain of human polo-like kinase 1

Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 A resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the kcat of the reaction, with little change in the apparent Km for the protein or nucleotide substrates (kcat = 0.0094, 0.0376, and 0.0049 s-1 and Km(ATP) = 3.2, 4.0, and 3.0 microM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.     

The crystal structure of the BAR domain from human Bin1/amphiphysin II and its implications for molecular recognition

BAR domains are found in proteins that bind and remodel membranes and participate in cytoskeletal and nuclear processes. Here, we report the crystal structure of the BAR domain from the human Bin1 protein at 2.0 A resolution. Both the quaternary and tertiary architectures of the homodimeric Bin1BAR domain are built upon "knobs-into-holes" packing of side chains, like those found in conventional left-handed coiled-coils, and this packing governs the curvature of a putative membrane-engaging concave face. Our calculations indicate that the Bin1BAR domain contains two potential sites for protein-protein interactions on the convex face of the dimer. Comparative analysis of structural features reveals that at least three architectural subtypes of the BAR domain are encoded in the human genome, represented by the Arfaptin, Bin1/Amphiphysin, and IRSp53 BAR domains. We discuss how these principal groups may differ in their potential to form regulatory heterotypic interactions.     

The crystal structure of human receptor protein tyrosine phosphatase kappa phosphatase domain 1

The receptor-type protein tyrosine phosphatases (RPTPs) are integral membrane proteins composed of extracellular adhesion molecule-like domains, a single transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain consists of tandem PTP domains, of which the D1 domain is enzymatically active. RPTPkappa is a member of the R2A/IIb subfamily of RPTPs along with RPTPmu, RPTPrho, and RPTPlambda. Here, we have determined the crystal structure of catalytically active, monomeric D1 domain of RPTPkappa at 1.9 A. Structural comparison with other PTP family members indicates an overall classical PTP architecture of twisted mixed beta-sheets flanked by alpha-helices, in which the catalytically important WPD loop is in an unhindered open conformation. Though the residues forming the dimeric interface in the RPTPmu structure are all conserved, they are not involved in the protein-protein interaction in RPTPkappa. The N-terminal beta-strand, formed by betax association with betay, is conserved only in RPTPs but not in cytosolic PTPs, and this feature is conserved in the RPTPkappa structure forming a beta-strand. Analytical ultracentrifugation studies show that the presence of reducing agents and higher ionic strength are necessary to maintain RPTPkappa as a monomer. In this family the crystal structure of catalytically active RPTPmu D1 was solved as a dimer, but the dimerization was proposed to be a consequence of crystallization since the protein was monomeric in solution. In agreement, we show that RPTPkappa is monomeric in solution and crystal structure.     

Non-proteinogenic amino acids in the pThr-2 position of a pentamer peptide that confer high binding affinity for the polo box domain (PBD) of polo-like kinase 1 (Plk1)

We report herein that incorporating long-chain alkylphenyl-containing non-proteinogenic amino acids in place of His at the pT-2 position of the parent polo-like kinase 1 (Plk1) polo box domain (PBD)-binding pentapeptide, PLHSpT (1a) increases affinity. For certain analogs, approximately two orders-of-magnitude improvement in affinity was observed. Although, none of the new analogs was as potent as our previously described peptide 1b, in which the pT-2 histidine imidazole ring is alkylated at its π nitrogen (N3), our current finding that the isomeric His(N1)-analog (1c) binds with approximately 50-fold less affinity than 1b, indicates the positional importance of attachment to the His imidazole ring. Our demonstration that a range of modified residues at the pT-2 position can enhance binding affinity, should facilitate the development of minimally-sized Plk1 PBD-binding antagonists.     

Solution structure of the ubiquitin-associated domain of human BMSC-UbP and its complex with ubiquitin

Ubiquitin is an important cellular signal that targets proteins for degradation or regulates their functions. The previously identified BMSC-UbP protein derived from bone marrow stromal cells contains a ubiquitin-associated (UBA) domain at the C terminus that has been implicated in linking cellular processes and the ubiquitin system. Here, we report the solution NMR structure of the UBA domain of human BMSC-UbP protein and its complex with ubiquitin. The structure determination was facilitated by using a solubility-enhancement tag (SET) GB1, immunoglobulin G binding domain 1 of Streptococcal protein G. The results show that BMSC-UbP UBA domain is primarily comprised of three alpha-helices with a hydrophobic patch defined by residues within the C terminus of helix-1, loop-1, and helix-3. The M-G-I motif is similar to the M/L-G-F/Y motifs conserved in most UBA domains. Chemical shift perturbation study revealed that the UBA domain binds with the conserved five-stranded beta-sheet of ubiquitin via hydrophobic interactions with the dissociation constant (KD) of approximately 17 microM. The structural model of BMSC-UbP UBA domain complexed with ubiquitin was constructed by chemical shift mapping combined with the program HADDOCK, which is in agreement with the result from mutagenesis studies. In the complex structure, three residues (Met76, Ile78, and Leu99) of BMSC-UbP UBA form a trident anchoring the domain to the hydrophobic concave surface of ubiquitin defined by residues Leu8, Ile44, His68, and Val70. This complex structure may provide clues for BMSC-UbP functions and structural insights into the UBA domains of other ubiquitin-associated proteins that share high sequence homology with BMSC-UbP UBA domain.     

The 2.7 A crystal structure of the autoinhibited human c-Fms kinase domain

c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine kinases (RTKs), is the receptor for macrophage colony stimulating factor (CSF-1) that regulates proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Abnormal expression of c-fms proto-oncogene is associated with a significant number of human pathologies, including a variety of cancers and rheumatoid arthritis. Accordingly, c-Fms represents an attractive therapeutic target. To further understand the regulation of c-Fms, we determined the 2.7 A resolution crystal structure of the cytosolic domain of c-Fms that comprised the kinase domain and the juxtamembrane domain. The structure reveals the crucial inhibitory role of the juxtamembrane domain (JM) that binds to a hydrophobic site immediately adjacent to the ATP binding pocket. This interaction prevents the activation loop from adopting an active conformation thereby locking the c-Fms kinase into an autoinhibited state. As observed for other members of the PDGF receptor family, namely c-Kit and Flt3, three JM-derived tyrosine residues primarily drive the mechanism for autoinhibition in c-Fms, therefore defining a common autoinhibitory mechanism within this family. Moreover the structure provides an understanding of c-Fms inhibition by Gleevec as well as providing a platform for the development of more selective inhibitors that target the inactive conformation of c-Fms kinase.     

Osteopontin localizes to the nucleus of 293 cells and associates with polo-like kinase-1

Osteopontin (OPN) is a secreted phosphoprotein involved in cellular proliferation and associated with tumor progression. Although an intracellular form of OPN has been described, its function remains unknown. In this study, a novel nuclear location for intracellular OPN and a correlation with cell division were demonstrated. OPN distinctly localized to the nucleus in a subset of transiently transfected human embryonic kidney 293 cells. Immunoblotting confirmed the nuclear location of native OPN, and results from immunofluorescence studies suggested an association between nuclear OPN and cell cycle progression. Flow cytometry revealed that nuclear and cellular OPN content rose significantly during the S and G₂/M phases, respectively. Treatment of cells with the DNA polymerase inhibitor aphidicolin prevented cell cycling and greatly reduced cellular OPN content. The intracellular location of OPN coincided with polo-like kinase-1 (Plk-1), a member of the polo-like kinase family, which, in part through their regulation of centrosome-related events, are integral to successful cellular mitosis. OPN and Plk-1 were coimmunoprecipitated from nuclear, but not cystoslic, extracts, demonstrating an interaction that is limited to the nucleus, presumably during mitosis. Deletion of the COOH terminus of OPN militated against nuclear localization and Plk-1 interaction. Elevated expression of OPN was also associated with an increase in the number of multinucleate 293 cells, whereas transfection of the COOH-terminal-deleted OPN decreased the percentage of multinucleate cells below basal levels. These findings implicate intranuclear OPN as a participant in the process of cell duplication. cell cycle; spindle; nuclear     

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein

Background: Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition.Results: The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 Å resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel α/β fold and contains a Zn²⁺ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (α1¹–loop12–α2¹; where ¹ represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23–α3¹–loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7.Conclusions: The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.     

The X-ray crystal structure of human gamma S-crystallin C-terminal domain.

gammaS-crystallin is a major human lens protein found in the outer region of the eye lens, where the refractive index is low. Because crystallins are not renewed they acquire post-translational modifications that may perturb stability and solubility. In common with other members of the betagamma-crystallin superfamily, gammaS-crystallin comprises two similar beta-sheet domains. The crystal structure of the C-terminal domain of human gammaS-crystallin has been solved at 2.4 A resolution. The structure shows that in the in vitro expressed protein, the buried cysteines remain reduced. The backbone conformation of the "tyrosine corner" differs from that of other betagamma-crystallins because of deviation from the consensus sequence. The two C-terminal domains in the asymmetric unit are organized about a slightly distorted 2-fold axis to form a dimer with similar geometry to full-length two-domain family members. Two glutamines found in lattice contacts may be important for short range interactions in the lens. An asparagine known to be deamidated in human cataract is located in a highly ordered structural region.     

The crystal structure of human quinolinic acid phosphoribosyltransferase in complex with its inhibitor phthalic acid

Quinolinic acid (QA), a biologically potent but neurodestructive metabolite is catabolized by quinolinic acid phosphoribosyltransferase (QPRT) in the first step of the de novo NAD⁺ biosynthesis pathway. This puts QPRT at the junction of two different pathways, that is, de novo NAD⁺ biosynthesis and the kynurenine pathway of tryptophan degradation. Thus, QPRT is an important enzyme in terms of its biological impact and its potential as a therapeutic target. Here, we report the crystal structure of human QPRT bound to its inhibitor phthalic acid (PHT) and kinetic analysis of PHT inhibition of human QPRT. This structure, determined at 2.55 Å resolution, shows an elaborate hydrogen bonding network that helps in recognition of PHT and consequently its substrate QA. In addition to this hydrogen bonding network, we observe extensive van der Waals contacts with the PHT ring that might be important for correctly orientating the substrate QA during catalysis. Moreover, our crystal form allows us to observe an intact hexamer in both the apo‐ and PHT‐bound forms in the same crystal system, which provides a direct comparison of unique subunit interfaces formed in hexameric human QPRT. We call these interfaces “nondimeric interfaces” to distinguish them from the typical dimeric interfaces observed in all QPRTs. We observe significant changes in the nondimeric interfaces in the QPRT hexamer upon binding PHT. Thus, the new structural and functional features of this enzyme we describe here will aid in understanding the function of hexameric QPRTs, which includes all eukaryotic and select prokaryotic QPRTs. Proteins 2014; 82:405–414. © 2013 Wiley Periodicals, Inc.     

The polo box is required for multiple functions of Plx1 in mitosis

Polo-like kinases comprise a family of evolutionarily conserved serine/threonine protein kinases that play multiple roles in cell cycle regulation. In addition to the N-terminal catalytic domain, polo-like kinases have one or two highly conserved C-terminal non-catalytic regions, termed polo boxes. These motifs are required for targeting these kinases to subcellular mitotic structures. Here we report that kinase-dead Xenopus polo-like kinase (Plx1NA) functions as a competitor of endogenous Plx1 for polo box binding site(s) and inhibits the activation of Cdc25C and the G(2)-M transition in vivo. However, kinase-dead Plx1 with a point mutation in the polo box region (Plx1NAWF) did not have inhibitory effects. The ability of Plx1NA to block activation of the anaphase-promoting complex/cyclosome also requires an intact polo box. Microinjection of Plx1NA but not Plx1NAWF mRNA into Xenopus embryos caused cleavage arrest and formation of monopolar spindles, an effect previously seen in embryos injected with anti-Plx1 antibody. Spindle assembly experiments in vitro also showed that only monopolar spindles formed in Xenopus egg extracts supplemented with recombinant Plx1NA and that the spindle assembly process was delayed. Taken together, these results indicate that the polo box is required for Plx1 function in both the G(2)-M and the metaphase/anaphase transitions during the cell cycle.     

The crystal structure of the SV40 T-antigen origin binding domain in complex with DNA

DNA replication is initiated upon binding of “initiators” to origins of replication. In simian virus 40 (SV40), the core origin contains four pentanucleotide binding sites organized as pairs of inverted repeats. Here we describe the crystal structures of the origin binding domain (obd) of the SV40 large T-antigen (T-ag) both with and without a subfragment of origin-containing DNA. In the co-structure, two T-ag obds are oriented in a head-to-head fashion on the same face of the DNA, and each T-ag obd engages the major groove. Although the obds are very close to each other when bound to this DNA target, they do not contact one another. These data provide a high-resolution structural model that explains site-specific binding to the origin and suggests how these interactions help direct the oligomerization events that culminate in assembly of the helicase-active dodecameric complex of T-ag.     

Cutting edge: molecular structure of the IL-1R-associated kinase-4 death domain and its implications for TLR signaling

IL-1R-associated kinase (IRAK) 4 is an essential component of innate immunity. IRAK-4 deficiency in mice and humans results in severe impairment of IL-1 and TLR signaling. We have solved the crystal structure for the death domain of Mus musculus IRAK-4 to 1.7 A resolution. This is the first glimpse of the structural details of a mammalian IRAK family member. The crystal structure reveals a six-helical bundle with a prominent loop, which among IRAKs and Pelle, a Drosophila homologue, is unique to IRAK-4. This highly structured loop contained between helices two and three, comprises an 11-aa stretch. Although innate immune domain recognition is thought to be very similar between Drosophila and mammals, this structural component points to a drastic difference. This structure can be used as a framework for future mutation and deletion studies and potential drug design.     

The crystal structure of the carboxy-terminal domain of human translation initiation factor eIF5

The carboxy-terminal domain (CTD) of eukaryotic initiation factor 5 (eIF5) plays a central role in the formation of the multifactor complex (MFC), an important intermediate for the 43 S pre-initiation complex assembly. The IF5-CTD interacts directly with the translation initiation factors eIF1, eIF2-beta, and eIF3c, thus forming together with eIF2 bound Met-tRNA(i)(Met) the MFC. In this work we present the high resolution crystal structure of eIF5-CTD. This domain of the protein is exclusively composed out of alpha-helices and is homologous to the carboxy-terminal domain of eIF2B-epsilon (eIF2Bepsilon-CTD). The most striking difference in the two structures is an additional carboxy-terminal helix in eIF5. The binding sites of eIF2-beta, eIF3 and eIF1 were mapped onto the structure. eIF2-beta and eIF3 bind to non-overlapping patches of negative and positive electrostatic potential, respectively.     

The crystal structure of the human Mov34 MPN domain reveals a metal-free dimer

The 26S proteasome is a large protein complex involved in protein degradation. We have shown previously that the PSMD7/Mov34 subunit of the human proteasome contains a proteolytically resistant MPN domain. MPN domain family members comprise subunits of the proteasome, COP9-signalosome and translation initiation factor 3 complexes. Here, the crystal structure of two C-terminally truncated proteins, MPN 1-186 and MPN 1-177, were solved to 1.96 and 3.0 A resolution, respectively. MPN 1-186 is formed by nine beta-strands surrounded by three alpha-helices plus a fourth alpha-helix at the C terminus. This final alpha-helix emerges from the domain core and folds along with a symmetrically related subunit, typical of a domain swap. The crystallographic dimer is consistent with size-exclusion chromatography and DLS analysis showing that MPN 1-186 is a dimer in solution. MPN 1-186 shows an overall architecture highly similar to the previously reported crystal structure of the Archaeal MPN domain AfJAMM of Archaeoglobus fulgidus. However, previous structural and biophysical analyses have shown that neither MPN 1-186 nor full-length human Mov34 bind metal, in opposition to the zinc-binding AfJAMM structures. The zinc ligand residues observed in AfJAMM are conserved in the yeast Rpn11 proteasome and Csn5 COP-signalosome subunits, which is consistent with the isopeptidase activity described for these proteins. The results presented here show that, although the MPN domain of Mov34 shows a typical metalloprotease fold, it is unable to coordinate a metal ion. This finding and amino acid sequence comparisons can explain why the MPN-containing proteins Mov34/PSMD7, RPN8, Csn6, Prp8p and the translation initiation factor 3 subunits f and h do not show catalytic isopeptidase activity, allowing us to propose the hypothesis that in these proteins the MPN domain has a primarily structural function.     

The crystal structure of the 1:1 inclusion complex of beta-cyclodextrin with benzamide

The 1:1 inclusion complex of beta-cyclodextrin and benzamide was prepared and characterized by single crystal X-ray diffraction, PXRD, TGA, and IR. This complex crystallizes in the monoclinic P2(1) space group with unit cell constants a=15.4244(16), b=10.1574(11), c=20.557(2)A, beta=110.074(2) degrees , V=3025.1(6)A(3). The guest molecule projects into the beta-cyclodextrin cavity from the primary hydroxyl side. The amide group protrudes from the primary hydroxyl side and forms hydrogen bonds with the adjacent beta-cyclodextrin molecule. There are six crystallized water molecules, which play crucial roles in crystal packing.     

The crystal structure of the 1:1 complex of beta-cyclodextrin with trans-cinnamic acid

The crystal structure of the 1:1 complex of beta-cyclodextrin (cyclomaltoheptaose) with trans-cinnamic acid was studied by X-ray diffraction. Two beta-cyclodextrin molecules related by a twofold crystal axis form dimers in the hydrophobic cavity of which, two guest molecules are entirely buried. The complex crystallizes in the monoclinic C2 space group with channel-type molecular packing. The oxygen atoms of the carboxylate group of the trans-cinnamic acid molecule form strong hydrogen bonds with two water molecules lying in the interdimeric space of the hydrophobic channel.