Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase.

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

Chromophore carbonyl twisting in fluorescent biosensors encodes direct readout of protein conformations with multicolor switching.

Fluorescent labeling of proteins is a powerful tool for probing structure-function relationships with many biosensing applications. Structure-based rules for systematically designing fluorescent biosensors require understanding ligand-mediated fluorescent response mechanisms which can be challenging to establish. We installed thiol-reactive derivatives of the naphthalene-based fluorophore Prodan into bacterial periplasmic glucose-binding proteins. Glucose binding elicited paired color exchanges in the excited and ground states of these conjugates. X-ray structures and mutagenesis studies established that glucose-mediated color switching arises from steric interactions that couple protein conformational changes to twisting of the Prodan carbonyl relative to its naphthalene plane. Mutations of residues contacting the carbonyl can optimize color switching by altering fluorophore conformational equilibria in the apo and glucose-bound proteins. A commonly accepted view is that Prodan derivatives report on protein conformations via solvatochromic effects due to changes in the dielectric of their local environment. Here we show that instead Prodan carbonyl twisting controls color switching. These insights enable structure-based biosensor design by coupling ligand-mediated protein conformational changes to internal chromophore twists through specific steric interactions between fluorophore and protein.

Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics.

DNA polymerases are essential for nucleic acid synthesis, cloning, sequencing and molecular diagnostics technologies. Conditional intein splicing is a powerful tool for controlling enzyme reactions. We have engineered a thermal switch into thermostable DNA polymerases from two structurally distinct polymerase families by inserting a thermally activated intein domain into a surface loop that is integral to the polymerase active site, thereby blocking DNA or RNA template access. The fusion proteins are inactive, but retain their structures, such that the intein excises during a heat pulse delivered at 70-80°C to generate spliced, active polymerases. This straightforward thermal activation step provides a highly effective, one-component 'hot-start' control of PCR reactions that enables accurate target amplification by minimizing unwanted by-products generated by off-target reactions. In one engineered enzyme, derived from Thermus aquaticus DNA polymerase, both DNA polymerase and reverse transcriptase activities are controlled by the intein, enabling single-reagent amplification of DNA and RNA under hot-start conditions. This engineered polymerase provides high-sensitivity detection for molecular diagnostics applications, amplifying 5-6 copies of the tested DNA and RNA targets with >95% certainty. The design principles used to engineer the inteins can be readily applied to construct other conditionally activated nucleic acid processing enzymes.

Structure-Guided Discovery of Potent Antifungals that Prevent Ras Signaling by Inhibiting Protein Farnesyltransferase.

Infections by fungal pathogens are difficult to treat due to a paucity of antifungals and emerging resistances. Next-generation antifungals therefore are needed urgently. We have developed compounds that prevent farnesylation of Cryptoccoccus neoformans Ras protein by inhibiting protein farnesyltransferase with 3-4 nanomolar affinities. Farnesylation directs Ras to the cell membrane and is required for infectivity of this lethal pathogenic fungus. Our high-affinity compounds inhibit fungal growth with 3-6 micromolar minimum inhibitory concentrations (MICs), 4- to 8-fold better than Fluconazole, an antifungal commonly used in the clinic. Compounds bound with distinct inhibition mechanisms at two alternative, partially overlapping binding sites, accessed via different inhibitor conformations. We showed that antifungal potency depends critically on the selected inhibition mechanism because this determines the efficacy of an inhibitor at low in vivo levels of enzyme and farnesyl substrate. We elucidated how chemical modifications of the antifungals encode desired inhibitor conformation and concomitant inhibitory mechanism.

Sensing and Processing of DNA Interstrand Crosslinks by the Mismatch Repair Pathway.

DNA interstrand crosslinks (ICLs) that are repaired in non-dividing cells must be recognized independently of replication-associated DNA unwinding. Using cell-free extracts from Xenopus eggs that support neither replication nor transcription, we establish that ICLs are recognized and processed by the mismatch repair (MMR) machinery. We find that ICL repair requires MutSα (MSH2-MSH6) and the mismatch recognition FXE motif in MSH6, strongly suggesting that MutSα functions as an ICL sensor. MutSα recruits MutLα and EXO1 to ICL lesions, and the catalytic activity of both these nucleases is essential for ICL repair. As anticipated for a DNA unwinding-independent recognition process, we demonstrate that least distorting ICLs fail to be recognized and repaired by the MMR machinery. This establishes that ICL structure is a critical determinant of repair efficiency outside of DNA replication.

Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I.

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity.

A Farnesyltransferase Acts to Inhibit Ectopic Neurite Formation in C. elegans.

Genetic pathways that regulate nascent neurite formation play a critical role in neuronal morphogenesis. The core planar cell polarity components VANG-1/Van Gogh and PRKL-1/Prickle are involved in blocking inappropriate neurite formation in a subset of motor neurons in C. elegans. A genetic screen for mutants that display supernumerary neurites was performed to identify additional factors involved in this process. This screen identified mutations in fntb-1, the ß subunit of farnesyltransferase. We show that fntb-1 is expressed in neurons and acts cell-autonomously to regulate neurite formation. Prickle proteins are known to be post-translationally modified by farnesylation at their C-terminal CAAX motifs. We show that PRKL-1 can be recruited to the plasma membrane in both a CAAX-dependent and CAAX-independent manner but that PRKL-1 can only inhibit neurite formation in a CAAX-dependent manner.

The Closing Mechanism of DNA Polymerase I at Atomic Resolution.

DNA polymerases must quickly and accurately distinguish between similar nucleic acids to form Watson-Crick base pairs and avoid DNA replication errors. Deoxynucleoside triphosphate (dNTP) binding to the DNA polymerase active site induces a large conformational change that is difficult to characterize experimentally on an atomic level. Here, we report an X-ray crystal structure of DNA polymerase I bound to DNA in the open conformation with a dNTP present in the active site. We use this structure to computationally simulate the open to closed transition of DNA polymerase in the presence of a Watson-Crick base pair. Our microsecond simulations allowed us to characterize the key steps involved in active site assembly, and propose the sequence of events involved in the prechemistry steps of DNA polymerase catalysis. They also reveal new features of the polymerase mechanism, such as a conserved histidine as a potential proton acceptor from the primer 3'-hydroxyl.

Rapid analysis of protein farnesyltransferase substrate specificity using peptide libraries and isoprenoid diphosphate analogues.

Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequent visualization with streptavidin-AP. Screening of the CVa2X and CCa2X libraries with Rattus norvegicus PFTase revealed reaction by many known recognition sequences as well as numerous unknown ones. Some of the latter occur in the genomes of bacteria and viruses and may be important for pathogenesis, suggesting new targets for therapeutic intervention. Screening of the CVa2X library with alkyne-functionalized isoprenoid substrates showed that those prepared from C10 or C15 precursors gave similar results, whereas the analogue synthesized from a C5 unit gave a different pattern of reactivity. Lastly, the substrate specificities of PFTases from three organisms (R. norvegicus, Saccharomyces cerevisiae, and Candida albicans) were compared using CVa2X libraries. R. norvegicus PFTase was found to share more peptide substrates with S. cerevisiae PFTase than with C. albicans PFTase. In general, this method is a highly efficient strategy for rapidly probing the specificity of this important enzyme.

Crystal structures of the fungal pathogen Aspergillus fumigatus protein farnesyltransferase complexed with substrates and inhibitors reveal features for antifungal drug design.

Species of the fungal genus Aspergillus are significant human and agricultural pathogens that are often refractory to existing antifungal treatments. Protein farnesyltransferase (FTase), a critical enzyme in eukaryotes, is an attractive potential target for antifungal drug discovery. We report high-resolution structures of A. fumigatus FTase (AfFTase) in complex with substrates and inhibitors. Comparison of structures with farnesyldiphosphate (FPP) bound in the absence or presence of peptide substrate, corresponding to successive steps in ordered substrate binding, revealed that the second substrate-binding step is accompanied by motions of a loop in the catalytic site. Re-examination of other FTase structures showed that this motion is conserved. The substrate- and product-binding clefts in the AfFTase active site are wider than in human FTase (hFTase). Widening is a consequence of small shifts in the α-helices that comprise the majority of the FTase structure, which in turn arise from sequence variation in the hydrophobic core of the protein. These structural effects are key features that distinguish fungal FTases from hFTase. Their variation results in differences in steady-state enzyme kinetics and inhibitor interactions and presents opportunities for developing selective anti-fungal drugs by exploiting size differences in the active sites. We illustrate the latter by comparing the interaction of ED5 and Tipifarnib with hFTase and AfFTase. In AfFTase, the wider groove enables ED5 to bind in the presence of FPP, whereas in hFTase it binds only in the absence of substrate. Tipifarnib binds similarly to both enzymes but makes less extensive contacts in AfFTase with consequently weaker binding.

Visualization of synaptic inhibition with an optogenetic sensor developed by cell-free protein engineering automation.

We describe an engineered fluorescent optogenetic sensor, SuperClomeleon, that robustly detects inhibitory synaptic activity in single, cultured mouse neurons by reporting intracellular chloride changes produced by exogenous GABA or inhibitory synaptic activity. Using a cell-free protein engineering automation methodology that bypasses gene cloning, we iteratively constructed, produced, and assayed hundreds of mutations in binding-site residues to identify improvements in Clomeleon, a first-generation, suboptimal sensor. Structural analysis revealed that these improvements involve halide contacts and distant side chain rearrangements. The development of optogenetic sensors that respond to neural activity enables cellular tracking of neural activity using optical, rather than electrophysiological, signals. Construction of such sensors using in vitro protein engineering establishes a powerful approach for developing new probes for brain imaging.

Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides.

In addition to discriminating against base pair mismatches, DNA polymerases exhibit a high degree of selectivity for deoxyribonucleotides over ribo- or dideoxynucleotides. It has been proposed that a single active site residue (steric gate) blocks productive binding of nucleotides containing 2'-hydroxyls. Although this steric gate plays a role in sugar moiety discrimination, its interactions do not account fully for the observed behavior of mutants. Here we present 10 high resolution crystal structures and enzyme kinetic analyses of Bacillus DNA polymerase I large fragment variants complexed with deoxy-, ribo-, and dideoxynucleotides and a DNA substrate. Taken together, these data present a more nuanced and general mechanism for nucleotide discrimination in which ensembles of intermediate conformations in the active site trap non-cognate substrates. It is known that the active site O-helix transitions from an open state in the absence of nucleotide substrates to a ternary complex closed state in which the reactive groups are aligned for catalysis. Substrate misalignment in the closed state plays a fundamental part in preventing non-cognate nucleotide misincorpation. The structures presented here show that additional O-helix conformations intermediate between the open and closed state extremes create an ensemble of binding sites that trap and misalign non-cognate nucleotides. Water-mediated interactions, absent in the fully closed state, play an important role in formation of these binding sites and can be remodeled to accommodate different non-cognate substrates. This mechanism may extend also to base pair discrimination.

Covalent protein-oligonucleotide conjugates by copper-free click reaction.

Covalent protein-oligodeoxynucleotide (protein-ODN) conjugates are useful in a number of biological applications, but synthesizing discrete conjugates-where the connection between the two components is at a defined location in both the protein and the ODN-under mild conditions with significant yield can be a challenge. In this article, we demonstrate a strategy for synthesizing discrete protein-ODN conjugates using strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC, a copper-free 'click' reaction). Azide-functionalized proteins, prepared by enzymatic prenylation of C-terminal CVIA tags with synthetic azidoprenyl diphosphates, were 'clicked' to ODNs that had been modified with a strained dibenzocyclooctyne (DIBO-ODN). The resulting protein-ODN conjugates were purified and characterized by size-exclusion chromatography and gel electrophoresis. We find that the yields and reaction times of the SPAAC bioconjugation reactions are comparable to those previously reported for copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) bioconjugation, but require no catalyst. The same SPAAC chemistry was used to immobilize azide-modified proteins onto surfaces, using surface-bound DIBO-ODN as a heterobifunctional linker. Cu-free click bioconjugation of proteins to ODNs is a simple and versatile alternative to Cu-catalyzed click methods.

Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis.

Even though high-fidelity polymerases copy DNA with remarkable accuracy, some base-pair mismatches are incorporated at low frequency, leading to spontaneous mutagenesis. Using high-resolution X-ray crystallographic analysis of a DNA polymerase that catalyzes replication in crystals, we observe that a C • A mismatch can mimic the shape of cognate base pairs at the site of incorporation. This shape mimicry enables the mismatch to evade the error detection mechanisms of the polymerase, which would normally either prevent mismatch incorporation or promote its nucleolytic excision. Movement of a single proton on one of the mismatched bases alters the hydrogen-bonding pattern such that a base pair forms with an overall shape that is virtually indistinguishable from a canonical, Watson-Crick base pair in double-stranded DNA. These observations provide structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis, a long-standing concept that has been difficult to demonstrate directly.

Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutSß.

MutSß is a eukaryotic mismatch repair protein that preferentially targets extrahelical unpaired nucleotides and shares partial functional redundancy with MutSα (MSH2-MSH6). Although mismatch recognition by MutSα has been shown to involve a conserved Phe-X-Glu motif, little is known about the lesion-binding mechanism of MutSß. Combined MSH3/MSH6 deficiency triggers a strong predisposition to cancer in mice and defects in msh2 and msh6 account for roughly half of hereditary nonpolyposis colorectal cancer mutations. These three MutS homologs are also believed to play a role in trinucleotide repeat instability, which is a hallmark of many neurodegenerative disorders. The baculovirus overexpression and purification of recombinant human MutSß and three truncation mutants are presented here. Binding assays with heteroduplex DNA were carried out for biochemical characterization. Crystallization and preliminary X-ray diffraction analysis of the protein bound to a heteroduplex DNA substrate are also reported.

Structures of Cryptococcus neoformans protein farnesyltransferase reveal strategies for developing inhibitors that target fungal pathogens.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

The structure of a high fidelity DNA polymerase bound to a mismatched nucleotide reveals an "ajar" intermediate conformation in the nucleotide selection mechanism.

To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG:dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established "open" and "closed" states. In this "ajar" conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation.

Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family.

Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5' structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. hExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5' ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exonucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant solution to a complex geometrical puzzle of substrate recognition and processing.

Structure-based design and synthesis of potent, ethylenediamine-based, mammalian farnesyltransferase inhibitors as anticancer agents.

A potent class of anticancer, human farnesyltransferase (hFTase) inhibitors has been identified by "piggy-backing" on potent, antimalarial inhibitors of Plasmodium falciparum farnesyltransferase (PfFTase). On the basis of a 4-fold substituted ethylenediamine scaffold, the inhibitors are structurally simple and readily derivatized, facilitating the extensive structure-activity relationship (SAR) study reported herein. Our most potent inhibitor is compound 1f, which exhibited an in vitro hFTase IC(50) value of 25 nM and a whole cell H-Ras processing IC(50) value of 90 nM. Moreover, it is noteworthy that several of our inhibitors proved highly selective for hFTase (up to 333-fold) over the related prenyltransferase enzyme geranylgeranyltransferase-I (GGTase-I). A crystal structure of inhibitor 1a co-crystallized with farnesyl pyrophosphate (FPP) in the active site of rat FTase illustrates that the para-benzonitrile moiety of 1a is stabilized by a π-π stacking interaction with the Y361ß residue, suggesting a structural explanation for the observed importance of this component of our inhibitors.

The mechanism of the translocation step in DNA replication by DNA polymerase I: a computer simulation analysis.

High-fidelity DNA polymerases copy DNA rapidly and accurately by adding correct deoxynucleotide triphosphates to a growing primer strand of DNA. Following nucleotide incorporation, a series of conformational changes translocate the DNA substrate by one base pair step, readying the polymerase for the next round of incorporation. Molecular dynamics simulations indicate that the translocation consists globally of a polymerase fingers-opening transition, followed by the DNA displacement and the insertion of the template base into the preinsertion site. They also show that the pyrophosphate release facilitates the opening transition and that the universally conserved Y714 plays a key role in coupling polymerase opening to DNA translocation. The transition involves several metastable intermediates in one of which the O helix is bent in the vicinity of G711. Completion of the translocation appears to require a gating motion of the O1 helix, perhaps facilitated by the presence of G715. These roles are consistent with the high level of conservation of Y714 and the two glycine residues at these positions. It is likely that a corresponding mechanism is applicable to other polymerases.