Engineering the product profile of a polysialyltransferase.

Oligo- and polysaccharides have myriad applications as therapeutic reagents from glycoconjugate vaccines to matrices for tissue engineering. Polysaccharide length may vary over several orders of magnitude and is a critical determinant of both their physical properties and biological activities. Therefore, the tailored synthesis of oligo- and polysaccharides of defined size is a major goal for glycoengineering. By mutagenesis and screening of a bacterial polysialyltransferase (polyST), we identified a single-residue switch that controls the size distribution of polymeric products. Specific substitutions at this site yielded distributive enzymes that synthesize polysaccharides with narrow size distribution ideal for glycoengineering applications. Mechanistic investigation revealed that the wild-type enzyme has an extended binding site that accommodates at least 20 residues of the growing polymer; changes in affinity along this binding site allow fine-tuning of the enzyme's product distribution.

Functional consequences of mutating conserved SF2 helicase motifs in the Type III restriction endonuclease EcoP15I translocase domain.

For efficient DNA hydrolysis, Type III restriction endonuclease EcoP15I interacts with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of two methylation (Mod) subunits and a single restriction (Res) subunit yielding a multifunctional enzyme complex able to methylate or to hydrolyse DNA. Comprehensive sequence alignments, limited proteolysis and mass spectroscopy suggested that the Res subunit is a fusion of a motor or translocase (Tr) domain of superfamily II helicases and an endonuclease domain with a catalytic PD…EXK motif. In the Tr domain, seven predicted helicase motifs (I, Ia, II-VI), a recently discovered Q-tip motif and three additional regions (IIIa, IVa, Va) conserved among Type III restriction enzymes have been identified that are predicted to be involved in DNA binding and ATP hydrolysis. Because DNA unwinding activity for EcoP15I (as for bona fide helicases) has never been found and EcoP15I ATPase rates are only low, the functional importance of the helicase motifs and regions was questionable and has never been probed systematically. Therefore, we mutated all helicase motifs and conserved regions predicted in Type III restriction enzyme EcoP15I and examined the functional consequences on EcoP15I enzyme activity and the structural integrity of the variants by CD spectroscopy. The resulting eleven enzyme variants all, except variant IVa, are properly folded showing the same secondary structure distribution as the wild-type enzyme. Classical helicase motifs I-VI are important for ATP and DNA cleavage by EcoP15I and mutations therein led to complete loss of ATPase and cleavage activity. Among the catalytically inactive enzyme variants three preserved the ability to bind ATP. In contrast, newly assigned motifs Q-tip, Ia and Va are not essential for EcoP15I activity and the corresponding enzyme variants were still catalytically active. DNA binding was only marginally reduced (2-7 fold) in all enzyme variants tested.

Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity.

For efficient DNA cleavage, the Type III restriction endonuclease EcoP15I communicates with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of methylation (Mod) and restriction (Res) subunits forming a multifunctional enzyme complex able to methylate or to cleave DNA. In this study, we determined by different analytical methods that EcoP15I contains a single Res subunit in a Mod(2)Res stoichiometry. The Res subunit comprises a translocase (Tr) domain carrying functional motifs of superfamily 2 helicases and an endonuclease domain with a PD..D/EXK motif. We show that the isolated Tr domain retains ATP-hydrolyzing activity and binds single- and double-stranded DNA in a sequence-independent manner. To localize the regions of DNA binding, we screened peptide arrays representing the entire Res sequence for their ability to interact with DNA. We discovered four DNA-binding regions in the Tr domain and two DNA-binding regions in the endonuclease domain. Modelling of the Tr domain shows that these multiple DNA-binding regions are located on the surface, free to interact with DNA. Interestingly, the positions of the DNA-binding regions are conserved among other Type III restriction endonucleases.

On the divalent metal ion dependence of DNA cleavage by restriction endonucleases of the EcoRI family.

Restriction endonucleases of the PD...D/EXK family need Mg(2+) for DNA cleavage. Whereas Mg(2+) (or Mn(2+)) promotes catalysis, Ca(2+) (without Mg(2+)) only supports DNA binding. The role of Mg(2+) in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg(2+) involved in catalysis. To address this problem, we measured the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me(2+) per active site. DNA cleavage experiments were carried out at various Mg(2+) and Mn(2+) concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg(2+) and Mn(2+) concentration dependence. In general, the Mg(2+) concentration optimum (between approximately 1 and 10 mM) is higher than the Mn(2+) concentration optimum (between approximately 0.1 and 1 mM). At still higher Mg(2+) or Mn(2+) concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca(2+). Based on these results, we propose that one Mg(2+) or Mn(2+) is critical for restriction enzyme activation, and binding of a second Me(2+) plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg(2+) or Mn(2+) mainly leads to an increase in K(m), such that the inhibitory effect of excess Mg(2+) or Mn(2+) can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me(2+) binding to these enzymes.

Molecular analysis of restriction endonuclease EcoRII from Escherichia coli reveals precise regulation of its enzymatic activity by autoinhibition.

Bacterial restriction endonuclease EcoRII requires two recognition sites to cleave DNA. Proteolysis of EcoRII revealed the existence of two stable domains, EcoRII-N and EcoRII-C. Reduction of the enzyme to its C-terminal domain, EcoRII-C, unleashed the enzyme activity; this truncated form no longer needed two recognition sites and cleaved DNA much more efficiently than EcoRII wild-type. The crystal structure of EcoRII showed that probably the N-terminal domain sterically occludes the catalytic site, thus apparently controlling the cleavage activity. Based on these data, EcoRII was the first restriction endonuclease for which an autoinhibition mechanism as regulatory strategy was proposed. In this study, we probed this assumption and searched for the inhibitory element that mediates autoinhibition. Here we show that repression of EcoRII-C is achieved by addition of the inhibitory domain EcoRII-N or by single soluble peptides thereof in trans. Moreover, we perturbed contacts between the N- and the C-terminal domain of EcoRII by site-directed mutagenesis and proved that beta-strand B1 and alpha-helix H2 are essential for autoinhibition; deletion of either secondary structural element completely relieved EcoRII autoinhibition. This potent regulation principle that keeps EcoRII enzyme activity controlled might protect bacteria against suicidal restriction of rare unmodified recognition sites in the cellular genome.

Cyclin E/Cdk2, P/CAF, and E1A regulate the transactivation of the c-myc promoter by FOXM1.

FOXM1c transactivates the c-myc promoter by binding directly to its TATA-boxes. The present study demonstrates that the transactivation of the c-myc promoter by FOXM1c is enhanced by the key proliferation signal cyclin E/Cdk2, but repressed by P/CAF and the adenoviral oncoprotein E1A. Furthermore, FOXM1c interacts with the coactivator and histone acetyltransferase P/CAF. This study shows that, on the c-myc-P1 TATA-box, FOXM1c does not function simply as normal transcription factor just binding to an unusual site. Moreover, the inhibitory N-terminus of FOXM1c does not inhibit its transrepression domain or its EDA. Others reported that a cyclin/Cdk-binding LXL-motif of the splice variant FoxM1b is required for its interaction with Cdk2, Cdk1, and p27, its phosphorylation by Cdk1 and its activation by Cdc25B. In contrast, we now demonstrate that this LXL-motif is not required for the activation of FOXM1c by cyclin D1/Cdk4, cyclin E/Cdk and cyclin A/Cdk2 or for the repression of FOXM1c by p27.

The c-myc promoter: still MysterY and challenge.

The transcription factor c-Myc is a key regulator of cell proliferation, cell growth, differentiation, and apoptosis. Deregulated c-myc expression possesses a high transformation potential and the proto-oncogene c-myc represents a promising target in anticancer therapy. This review on the c-myc promoter describes its organization, the different levels of its normal regulation (including initiation and elongation of transcription, the dual P1/P2 promoters, chromatin structure, c-Myc autosuppression) as well as its deregulation in Burkitt's lymphoma. Furthermore, it summarizes the many different transcription factors, signal transduction pathways, and feedback loops that activate or repress c-myc transcription. Finally, a concept for regulation of the c-myc promoter in different biological settings, for example, immediate-early induction, constant expression throughout the cell cycle in continuously cycling cells, repression during terminal differentiation and deregulation in cancer, is formulated.

FOXM1, a typical proliferation-associated transcription factor.

FOXM1 is a typical proliferation-associated transcription factor: it stimulates proliferation by promoting S-phase entry as well as M-phase entry and is involved in proper execution of mitosis. Accordingly, FOXM1 regulates genes that control G1/S-transition, S-phase progression, G2/M-transition and M-phase progression. Consistently, its expression and its activity are antagonistically regulated by many important proliferation and anti-proliferation signals. Furthermore, FOXM1 is implicated in tumorigenesis and contributes to both tumor initiation and progression. In addition to its function as a conventional transcription factor, FOXM1 transactivates the human c-myc P1 and P2 promoters directly via their TATA-boxes by a new transactivation mechanism, which it also employs for transactivation of the human c-fos, hsp70 and histone H2B/a promoters. This review summarizes the current knowledge on FOXM1, in particular its two different transactivation mechanisms, the regulation of its transcriptional activity by proliferation versus anti-proliferation signals and its function in normal cell cycle progression and tumorigenesis.

The central domain of transcription factor FOXM1c directly interacts with itself in vivo and switches from an essential to an inhibitory domain depending on the FOXM1c binding site.

We have previously shown that FOXM1c can transactivate its target genes by two different mechanisms, depending on the FOXM1c binding site. In the present study, by introducing a small 46-aa deletion, we confirm that the central domain of FOXM1c is essential for transactivation of the minimal c-myc P1 and P2 promoters via their TATA boxes, but functions as an inhibitory domain on conventional FOXM1c binding sites. Thus, distinct FOXM1c binding sites determine opposite functions of the central domain, suggesting allosteric control of its conformation by the DNA binding site. This is strongly supported by the identification of a direct in vivo interaction of the central domain with itself in the present study. In contrast, the DNA binding domain binds neither to itself nor to any other domain of FOXM1c. Transrepression by the central domain is unlikely to be achieved by recruitment of co-repressors, but instead seems to be mediated by direct interference with the basal transcription complex. Direct binding of the central domain to itself should be involved in transrepression. Finally, FOXM1c transactivates the chicken mim-1 promoter, whose TATA box represents a conventional FOXM1c binding site, so that transactivation follows neither of the above two mechanisms, but shows intermediate behavior.

FOXM1c and Sp1 transactivate the P1 and P2 promoters of human c-myc synergistically.

We have previously shown that FOXM1c transactivates the c-myc P1 and P2 promoters via their TATA-boxes by a new transactivation mechanism, namely by directly binding to the P1 and P2 TATA-boxes and to TBP, TFIIA, and TFIIB. We now confirm this surprising mechanism by demonstrating that FOXM1c transactivates the human c-myc P1 and P2 promoters synergistically with Sp1, a transcription factor known to bind and transactivate these two promoters. This synergism requires the P1 or P2 TATA-boxes as well as the respective Sp1-binding sites. Moreover FOXM1c binds directly to Sp1. Cooperative DNA binding, if it should occur, is not sufficient for synergism of Sp1 and FOXM1c at P1, but their contacts to multiple components of the basal transcription complex (TFIID, TFIIA, TFIIB) seem to be essential. However, FOXM1c does not synergize with Sp1 if it transactivates via its conventional binding site.

FOXM1c transactivates the human c-myc promoter directly via the two TATA boxes P1 and P2.

FOXM1c transactivates the c-myc promoter via the P1 and P2 TATA boxes using a new mechanism. Whereas the P1 TATA box TATAATGC requires its sequence context to be FOXM1c responsive, the P2 TATA box TATAAAAG alone is sufficient to confer FOXM1c responsiveness to any minimal promoter. FOXM1c transactivates by binding to the TATA box as well as directly to TATA-binding protein, transcription factor IIB and transcription factor IIA. This new transactivation mechanism is clearly distinguished from the function of FOXM1c as a conventional transcription factor. The central domain of FOXM1c functions as an essential domain for activation via the TATA box, but as an inhibitory domain (retinoblastoma protein-independent transrepression domain and retinoblastoma protein-recruiting negative regulatory domain) for transactivation via conventional FOXM1c-binding sites. Each promoter with the P2 TATA box TATAAAAG is postulated to be transactivated by FOXM1c. This was demonstrated for the promoters of c-fos, hsp70 and histone H2B/a. A database search revealed almost 300 probable FOXM1c target genes, many of which function in proliferation and tumorigenesis. Accordingly, dominant-negative FOXM1c proteins reduced cell growth approximately threefold, demonstrating a proliferation-stimulating function for wild-type FOXM1c.

FOXM1c is activated by cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1, but repressed by GSK-3alpha.

Two different inhibitory domains, N-terminus and central domain, keep FOXM1c almost inactive despite its strong transactivation domain. Here, we demonstrate that cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1 activate FOXM1c. Cyclin E/Cdk2 does not target its transactivation domain or its DNA-binding domain. Instead, its activating effect strictly depends on the presence of either the central domain or the N-terminus of FOXM1c and thus is completely lost if both inhibitory domains are deleted. Cyclin E/Cdk2 activates FOXM1c by releasing its transactivation domain from the repression by these two inhibitory domains. However, it does not directly increase the transactivation potential of the TAD. The DNA-binding is not affected by cyclin E/Cdk2, neither directly nor indirectly. These two activating effects of cyclin E/Cdk2 via central domain and N-terminus are additive. Cyclin A/Cdk2 and cyclin A/Cdk1 show similar characteristics. GSK-3alpha, another proliferation-associated kinase, represses FOXM1c.

Transcription factor FOXM1c is repressed by RB and activated by cyclin D1/Cdk4.

The proliferation-stimulating transactivator FOXM1c (MPP2) is repressed by RB and activated by cyclin D1/Cdk4 and therefore behaves like E2F. Despite its strong transactivation domain, FOXM1c is kept almost inactive by two different inhibitory domains, the N-terminus and the central domain. The tumor suppressor RB binds directly to the central domain of FOXM1c and thereby indirectly represses the transactivation domain, so that the central domain of FOXM1c functions as an RB-recruiting negative-regulatory domain. Cyclin D1/Cdk4 releases FOXM1c from this repression by RB and from the repression by its own inhibitory N-terminus, thereby strongly activating FOXM1c. However, cyclin D1/Cdk4 does not directly affect the transactivation domain or the DNA-binding domain. By phosphorylation of RB, but not FOXM1c, cyclin D1/Cdk4 interrupts their direct interaction and thus abrogates the repression of FOXM1c by RB. Cyclin D1/Cdk4 also eliminates the inhibition of the transactivation domain by the N-terminus of FOXM1c, probably by interruption of their direct interaction. Consequently, the G1-phase proliferation signal cyclin D1/Cdk4 converts FOXM1c from an almost inactive form into a strong transactivator in G1-phase, i.e., just at the time point at which the transcriptional activity of FOXM1 is required for stimulation of the G1/S-transition.

Despite its strong transactivation domain, transcription factor FOXM1c is kept almost inactive by two different inhibitory domains.

FOXM1c (MPP2) is an activating transcription factor with several nuclear localization signals, a forkhead domain for DNA binding, and a very strong acidic transactivation domain. Despite its very strong transactivation domain, FOXM1c is kept almost inactive by two different independent inhibitory domains, the N-terminus and the central domain. The N-terminus as a specific negative-regulatory domain directly binds to and thus inhibits the transactivation domain completely. However, it lacks any transrepression potential. In contrast, the central domain functions as a strong RB-independent transrepression domain and as an RB-recruiting negative-regulatory domain. The N-terminus alone is sufficient to eliminate transactivation, while the central domain alone represses the transactivation domain only partially. This hierarchy of the two inhibitory domains offers the possibility to activate the almost inactive wild type in two steps in vitro: deletion of the N-terminus results in a strong transactivator, while additional deletion of the central domain in a very strong transactivator. We suggest that the very high potential of the transactivation domain has to be tightly controlled by these two inhibitory domains because FOXM1 stimulates proliferation by promoting G1/S transition, as well as G2/M transition, and because deregulation of such potent activators of proliferation can result in tumorigenesis.

A monomeric mutant of restriction endonuclease EcoRI nicks DNA without sequence specificity.

We have mutated the monomer-monomer interface of the restriction endonuclease EcoRI in order to destabilize the homodimer and to stabilize heterodimers. Mutations of Leu158 to charged amino acid residues result in strong destabilization of the dimer. The largest effect was detected for the L158D mutant which is monomeric even at higher concentrations. It unspecifically degrades DNA by cleaving both single strands independently every 15 nucleotides on the average. Although cleavage is reproducible, it is not determined by nucleotide sequence but by general properties like conformation or deformability as has been found for other unspecific nucleases. Mutations of Ile230, which is in direct contact with Leu158 of the other subunit, cause structural changes with the loss of about ten percent alpha-helix content, but interfere only marginally with homodimerization and double strand cleavage. Again the mutation to aspartate shows the strongest effects. Mixtures of single mutants, one containing aspartate at one of the two positions and the other lysine at the corresponding position, form heterodimers. These are mainly stabilized compared to the homodimers by re-establishment of the wild-type hydrophobic interaction at the not mutated residues while an interaction of aspartate and lysine seems energetically unfavorable in this structural context.

Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin.

Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.

Two carbohydrate binding sites in the H(CC)-domain of tetanus neurotoxin are required for toxicity.

Tetanus neurotoxin binds via its carboxyl-terminal H(C)-fragment selectively to neurons mediated by complex gangliosides. We investigated the lactose and sialic acid binding pockets of four recently discovered potential binding sites employing site-directed mutagenesis. Substitution of residues in the lactose binding pocket drastically decreased the binding of the H(C)-fragment to immobilized gangliosides and to rat brain synaptosomes as well as the inhibitory action of recombinant full length tetanus neurotoxin on exocytosis at peripheral nerves. The conserved motif of S(1287)XWY(1290) em leader G(1300) assisted by N1219, D1222, and H1271 within the lactose binding site comprises a typical sugar binding pocket, as also present, for example, in cholera toxin. Replacement of the main residue of the sialic acid binding site, R1226, again caused a dramatic decline in binding affinity and neurotoxicity. Since the structural integrity of the H(C)-fragment mutants was verified by circular dichroism and fluorescence spectroscopy, these data provide the first biochemical evidence that two carbohydrate interaction sites participate in the binding and uptake process of tetanus neurotoxin. The simultaneous binding of one ganglioside molecule to each of the two binding sites was demonstrated by mass spectroscopy studies, whereas ganglioside-mediated linkage of native tetanus neurotoxin molecules was ruled out by size exclusion chromatography. Hence, a subsequent displacement of one ganglioside by a glycoprotein receptor is discussed.

Importance of phosphate contacts for sequence recognition by EcoRI restriction enzyme.

We have studied the importance of charge and hydrogen-bonding potential of the phosphodiester backbone for binding and cleavage by EcoRI restriction endonuclease. We used 12-mer oligodeoxynucleotide substrates with single substitutions of phosphates by chiral methylphosphonates at each position of the recognition sequence -pGpApApTpTpCp-. Binding was moderately reduced between 4- and 400-fold more or less equally for the R(P) and S(P)-analogues mainly caused by missing charge interaction. The range of cleavage effects was much wider. Four substrates were not cleaved at all. At both flanking positions and in the purine half of the sequence up to the central position, cleavage was more impaired than binding and differences between R(P) and S(P) diastereomeres were more pronounced. These effects are easily interpreted by direct phosphate contacts seen in the crystal structure. For the effects of substitutions in the pyrimidine half of the recognition sequence, more indirect effects have to be discussed.

Arg(362) and Tyr(365) of the botulinum neurotoxin type a light chain are involved in transition state stabilization.

The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn(2+) is coordinated by His(222) and His(226) of the Zn(2+) binding motif HisGluXXHis and Glu(261), whereas Glu(223) coordinates the water molecule required for hydrolysis as the fourth ligand. Recent analysis of a cocrystal of the BoNT/B LC and its substrate synaptobrevin 2 suggested that Arg(362) and Tyr(365) of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu(350) which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg(362) and substitution of Tyr(365) with Phe resulted in 79- and 34-fold lower k(cat)/K(m) values, respectively. These changes were provoked by decreased catalytic rates (k(cat)) and not by alterations of ground state substrate binding as evidenced by largely unchanged K(d) and K(m) values. None of these mutations affected the overall secondary structure or zinc content of the LC. These findings suggest that the guanidino group of Arg(362) and the hydroxyl group of Tyr(365) together accomplish transition state stabilization as was proposed for thermolysin, being the prototypical member of the gluzincin superfamily of metalloproteases. Mutation of Glu(350) dramatically diminished the hydrolytic activity which must partly be attributed to an altered active site fine structure as demonstrated by an increased sensitivity toward heat-induced denaturing and a lower Zn(2+) binding affinity. Glu(350) apparently occupies a central position in the active site and presumably positions His(222) and Arg(362).

Unusual structural organization of the endocytic proteins AP180 and epsin 1.

Epsin and AP180/CALM are important endocytic accessory proteins that are believed to be involved in the formation of clathrin coats. Both proteins associate with phosphorylated membrane inositol lipids through their epsin N-terminal homology domains and with other components of the endocytic machinery through short peptide motifs in their carboxyl-terminal segments. Using hydrodynamic and spectroscopic methods, we demonstrate that the parts of epsin 1 and AP180 that are involved in protein-protein interactions behave as poorly structured flexible polypeptide chains with little or no conventional secondary structure. The predominant cytosolic forms of both proteins are monomers. Furthermore, we show that recombinant epsin 1, like AP180, drives in vitro assembly of clathrin cages. We conclude that the epsin N-terminal homology domain-containing proteins AP180/CALM and epsin 1 have a very similar molecular architecture that is designed for the rapid and efficient recruitment of the principal coat components clathrin and AP-2 at the sites of coated pit assembly.