Presence Of AMP-PNP Research Articles

Probing the Mechanisms Underlying the Transport of the Vinca Alkaloids by P-glycoprotein

The efficacy of many cancer drugs is hindered by P-glycoprotein (Pgp), a cellular pump that removes drugs from cells. To improve chemotherapy, drugs capable of evading Pgp must be developed. Despite similarities in structure, vinca alkaloids (VAs) show disparate Pgp-mediated efflux ratios. ATPase activity and binding affinity studies show at least two binding sites for the VAs: high- and low-affinity sites that stimulate and inhibit the ATPase activity rate, respectively. The affinity for ATP from the ATPase kinetics curve for vinblastine (VBL) at the high-affinity site was 2- and 9-fold higher than vinorelbine (VRL) and vincristine (VCR), respectively. Conversely, VBL had the highest Km (ATP) for the low-affinity site. The dissociation constants (KDs) determined by protein fluorescence quenching were in the order VBL < VRL< VCR. The order of the KDs was reversed at higher substrate concentrations. Acrylamide quenching of protein fluorescence indicate that the VAs, either at 10 µM or 150 µM, predominantly maintain Pgp in an open-outward conformation. When 3.2 mM AMPPNP was present, 10 µM of either VBL, VRL, or VCR cause Pgp to shift to an open-outward conformation, while 150 µM of the VAs shifted the conformation of Pgp to an intermediate orientation, between opened inward and open-outward. However, the conformational shift induced by saturating AMPPNP and VCR condition was less than either VBL or VRL in the presence of AMPPNP. At 150 µM, atomic force microscopy (AFM) revealed that the VAs shift Pgp population to a predominantly open-inward conformation. Additionally, STDD NMR studies revealed comparable groups in VBL, VRL, and VCR are in contact with the protein during binding. Our results, when coupled with VAs-microtubule structure-activity relationship studies, could lay the foundation for developing next-generation VAs that are effective as anti-tumor agents. A model that illustrates the intricate process of Pgp-mediated transport of the VAs is presented.

Visualization of conformational transition of GRP94 in solution.

GRP94, an ER paralog of the heat-shock protein 90 family, binds and hydrolyses ATP to chaperone the folding and maturation of its selected clients. Compared with other hsp90 proteins, the in-solution conformational dynamics of GRP94 along the ATP hydrolysis cycle are less understood, hindering our understanding of its chaperoning mechanism. Leveraging small-angle X-ray scattering, negative-staining EM, and hydrogen-deuterium exchange coupled mass-spec, here we show that in its apo form, ∼60% of mouse GRP94 (mGRP94) populates an "extended" conformation, whereas the rest exist in either "close V" or "twist V" like "compact" conformations. Different from other hsp90 proteins, the presence of AMPPNP only impacts the relative abundance of the two compact conformations, rather than shifting the equilibrium between the "extended" and "compact" conformations of mGRP94. HDX-MS study of apo, AMPPNP-bound, and ADP-bound mGRP94 suggests a conformational transition from "twist V" to "close V" upon ATP binding and a back transition from "close V" to "twist V" upon ATP hydrolysis. These results illustrate the dissimilarities of GRP94 in conformation transition during ATP hydrolysis from other hsp90 paralogs.

Ultrafast Force-Clamp Spectroscopy of Microtubule-Binding Proteins.

Optical trapping has been instrumental for deciphering translocation mechanisms of the force-generating cytoskeletal proteins. However, studies of the dynamic interactions between microtubules (MTs) and MT-associated proteins (MAPs) with no motor activity are lagging. Investigating the motility of MAPs that can diffuse along MT walls is a particular challenge for optical-trapping assays because thermally driven motions rely on weak and highly transient interactions. Three-bead, ultrafast force-clamp (UFFC) spectroscopy has the potential to resolve static and diffusive translocations of different MAPs with sub-millisecond temporal resolution and sub-nanometer spatial precision. In this report, we present detailed procedures for implementing UFFC, including setup of the optical instrument and feedback control, immobilization and functionalization of pedestal beads, and preparation of MT dumbbells. Example results for strong static interactions were generated using the Kinesin-7 motor CENP-E in the presence of AMP-PNP. Time resolution for MAP-MT interactions in the UFFC assay is limited by the MT dumbbell relaxation time, which is significantly longer than reported for analogous experiments using actin filaments. UFFC, however, provides a unique opportunity for quantitative studies on MAPs that glide along MTs under a dragging force, as illustrated using the kinetochore-associated Ska complex.

Structural Insights into Mdn1, an Essential AAA Protein Required for Ribosome Biogenesis

Mdn1 is an essential AAA (ATPase associated with various activities) protein that removes assembly factors from distinct precursors of the ribosomal 60S subunit. However, Mdn1's large size (∼5,000 amino acid [aa]) and its limited homology to other well-studied proteins have restricted our understanding of its remodeling function. Here, we present structures for S.pombe Mdn1 in the presence of AMPPNP at up to ∼4Å or ATP plus Rbin-1, a chemical inhibitor, at ∼8Åresolution. These data reveal that Mdn1's MIDAS domain is tethered to its ring-shaped AAA domain through an ∼20nm long structured linker and a flexible ∼500 aa Asp/Glu-rich motif. We find that the MIDAS domain, which also binds other ribosome-assembly factors, docks onto the AAA ring in a nucleotide state-specific manner. Together, our findings reveal how conformational changes in the AAA ring can be directly transmitted to the MIDAS domain and thereby drive the targeted release of assembly factors from ribosomal 60S-subunit precursors.

Large Rotation of the N-terminal Domain of Hsp90 Is Important for Interaction with Some but Not All Client Proteins

The 90-kDa heat shock protein (Hsp90) chaperones the late folding steps of many protein kinases, transcription factors, and a diverse set of other protein clients not related in sequence and structure. Hsp90's interaction with clients appears to be coupled to a series of conformational changes. How these conformational changes contribute to its chaperone activity is currently unclear. Using crosslinking, hydrogen exchange mass spectrometry, and fluorescence experiments, we demonstrate here that the N-terminal domain of Hsp90 rotates by approximately 180° as compared to the crystal structure of yeast Hsp90 in complex with Sba1 and AMPPNP. Surprisingly, Aha1 but not Sba1 suppresses this rotation in the presence of AMPPNP but not in its absence. A minimum length of the largely unstructured linker between N-terminal and middle domain is necessary for this rotation, and interfering with the rotation strongly affects the interaction with Aha1 and the intrinsic and Aha1-stimulated ATPase activity. Surprisingly, suppression of the rotation only affects the activity of some clients and does not compromise yeast viability.

Structural and Functional Basis for Lipid Synergy on the Activity of the Antibacterial Peptide ABC Transporter McjD

The lipid bilayer is a dynamic environment that consists of a mixture of lipids with different properties that regulate the function of membrane proteins; these lipids are either annular, masking the protein hydrophobic surface, or specific lipids, essential for protein function. In this study, using tandem mass spectrometry, we have identified specific lipids associated with the Escherichia coli ABC transporter McjD, which translocates the antibacterial peptide MccJ25. Using non-denaturing mass spectrometry, we show that McjD in complex with MccJ25 survives the gas phase. Partial delipidation of McjD resulted in reduced ATPase activity and thermostability as shown by circular dichroism, both of which could be restored upon addition of defined E. coli lipids. We have resolved a phosphatidylglycerol lipid associated with McjD at 3.4 Å resolution, whereas molecular dynamic simulations carried out in different lipid environments assessed the binding of specific lipids to McjD. Combined, our data show a synergistic effect of zwitterionic and negatively charged lipids on the activity of McjD; the zwitterionic lipids provide structural stability to McjD, whereas the negatively charged lipids are essential for its function.

The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease.

Lon is an essential, multitasking AAA+ protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon’s N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning.

Structures of the CDK12/CycK complex with AMP-PNP reveal a flexible C-terminal kinase extension important for ATP binding.

Cyclin-dependent kinase 12 (CDK12) promotes transcriptional elongation by phosphorylation of the RNA polymerase II C-terminal domain (CTD). Structure-function studies show that this activity is dependent on a C-terminal kinase extension, as well as the binding of cyclin K (CycK). To better define these interactions we determined the crystal structure of the human CDK12/CycK complex with and without the kinase extension in the presence of AMP-PNP. The structures revealed novel features for a CDK, including a large β4-β5 loop insertion that contributes to the N-lobe interaction with the cyclin. We also observed two different conformations of the C-terminal kinase extension that effectively open and close the ATP pocket. Most notably, bound AMP-PNP was only observed when trapped in the closed state. Truncation of this C-terminal structure also diminished AMP-PNP binding, as well as the catalytic activity of the CDK12/CycK complex. Further kinetic measurements showed that the full length CDK12/CycK complex was significantly more active than the two crystallised constructs suggesting a critical role for additional domains. Overall, these results demonstrate the intrinsic flexibility of the C-terminal extension in CDK12 and highlight its importance for both ATP binding and kinase activity.

Nonhydrolyzable ATP analog 5'-adenylyl-imidodiphosphate (AMP-PNP) does not inhibit ATP-dependent scanning of leader sequence of mRNA.

The objective of the present work was to determine whether it is possible to use a nonhydrolyzable analog of ATP (AMP-PNP) as an inhibitor of ATP-dependent scanning of the leader sequence of eukaryotic mRNA in translation initiation-. The formation of ribosomal 48S initiation complexes at the start codon of the capped mRNA leader sequence of rabbit β-globin mRNA was studied. The study was carried out in a system composed of individual components of translation initiation. The dependences of the efficiency of formation of 48S initiation complexes on ATP concentration and incubation time were obtained in the absence and presence of AMP-PNP. It was found that AMP-PNP did not affect the efficiency of formation of 48S initiation complexes in all cases under study. We conclude that the uncleavable analog of ATP, AMP-PNP, is not an inhibitor of translation initiation in eukaryotes.

Nucleotide-dependent displacement and dynamics of the α-1 helix in kinesin revealed by site-directed spin labeling EPR

In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal region of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening. The inter-helix distance distribution showed that 20% of the spins have a peak characteristic of 1.4–1.7nm separation, which is similar to what is predicted from the X-ray crystal structure, albeit 80% were beyond the sensitivity limit (>2.5nm) of the method. Upon MT binding, the fraction of kinesin exhibiting an inter-helix distance of 1.4–1.7nm in the presence of AMPPNP (a non-hydrolysable ATP analog) and ADP was 20% and 25%, respectively. In the absence of nucleotide, this fraction increased to 40–50%. These nucleotide-induced changes in the fraction of kinesin undergoing displacement of the α-1 helix were found to be related to the fraction in which the NL undocked from the motor core. It is therefore suggested that a shift in the α-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core.

Duplex Destabilization by Four Ribosomal DEAD-Box Proteins

DEAD-box proteins are believed to participate in the folding of RNA by destabilizing RNA secondary or tertiary structures. Although these proteins bind and hydrolyze ATP, the mechanism by which nucleotide hydrolysis is coupled to helix destabilization may vary among different DEAD-box proteins. To investigate their abilities to disrupt helices and couple ATP hydrolysis to unwinding, we assayed the Saccharomyces cerevisiae ribosomal DEAD-box proteins, Dbp3p, Dbp4p, Rok1p, and Rrp3p utilizing a series of RNA substrates containing a short duplex and either a 5' or 3' single-stranded region. All four proteins unwound a 10 bp helix in vitro in the presence of ATP; however, significant dissociation of longer helices was not observed. While Dbp3p did not require a single-stranded extension to disrupt a helix, the unwinding activities of Dbp4p, Rok1p, and Rrp3p were substantially stimulated by either a 5' or 3' single-stranded extension. Interestingly, these proteins showed a clear length dependency with 3' extensions that was not observed with 5' extensions, suggesting that they bind substrates with a preferred orientation. In the presence of AMPPNP or ADP, all four proteins displayed displacement activity suggesting that nucleotide binding is sufficient to facilitate duplex disruption. Further enhancement of the strand displacement rate in the presence of ATP was observed for only Dbp3p and Rrp3p.

Structure of AMP-PNP-bound vitamin B12 transporter BtuCD–F

The ATP-binding cassette (ABC) transporter BtuCD mediates the uptake of vitamin B(12) across the inner membrane of Escherichia coli. Previous structures have shown the conformations of apo states, but the transport mechanism has remained unclear. Here we report the 3.5 Å crystal structure of the transporter-binding protein complex BtuCD-BtuF (BtuCD-F) trapped in an β-γ-imidoadenosine 5'-phosphate (AMP-PNP)-bound intermediate state. Although the ABC domains (BtuD subunits) form the expected closed sandwich dimer, the membrane-spanning BtuC subunits adopt a new conformation, with the central translocation pathway sealed by a previously unrecognized cytoplasmic gate. A fully enclosed cavity is thus formed approximately halfway across the membrane. It is large enough to accommodate a vitamin B(12) molecule, and radioligand trapping showed that liposome-reconstituted BtuCD-F indeed contains bound B(12) in the presence of AMP-PNP. In combination with engineered disulphide crosslinking and functional assays, our data suggest an unexpected peristaltic transport mechanism that is distinct from those observed in other ABC transporters.

Conformational Dynamics, Structure & Substrate Protein Interactions of Hsp90 Chaperones

Hsp90 is generally thought to transition between an open, apo and closed, ‘ATP’ conformation. A third, ADP state has been observed with the E. coli Hsp90. Using a combination of SAXS, EM single particle and crosslinking methods, we find that that all three states are universally conserved between bacteria, yeast and human Hsp90s. However the equilibrium and kinetics of the transitions between sates differ significantly. To increase conformational diversity in the E coli chaperone, there are two apo conformations (an open and Grp94-like state) established by a pH-dependent titration of a specific histidine. We also show that the conformational states are functionally distinct. These data support a conserved multi-state chaperone cycle where the conformational equilibrium varies between species, implicating evolutionary tuning of the different states to meet the specific client protein and metabolic environment of an organism. Current efforts focus on client-Hsp90 and cochaperone-Hsp90 interactions. We show that a deletion mutant of staph nuclease (Δ131Δ) that is unfolded and non-aggregating provides a powerful model substrate for interrogating Hsp90-client interactions. NMR reveals a distinct ∼25 residue region (the most structured within Δ131Δ) that interacts strongly with Hsp90, resulting in dramatic shifts in Hsp90 conformation. SAXS and NMR identify a region of the Hsp90 MD as the primary site of Δ131Δ binding. Most importantly, FRET measurements demonstrate that this model substrate catalyzes Hsp90 closure in the presence of AMPPNP. Similar effects have now been observed for other clients and with other Hsp90s. The human mitochondrial Hsp90 TRAP1 has a remarkable temperature-dependent closure demonstrating that kinetic rather than thermodynamic control is critical for understanding Hsp90 function. Three-dimensional cryoEM reconstructions for Hop-Hsp90, Hsp70-Hop-Hsp90complexes are underway and will be discussed.

Allosteric Interactions of DNA and Nucleotides with S. cerevisiae RSC

RSC (remodel the structure of chromatin) is an essential chromatin remodeler of Saccharomyces cerevisiae that has been shown to have DNA translocase properties. We studied the DNA binding properties of a "trimeric minimal RSC" (RSCt) of the RSC chromatin remodeling complex and the effect of nucleotides on this interaction using fluorescence anisotropy. RSCt binds to 20 bp fluorescein-labeled double-stranded DNA with a K(d) of ∼100 nM. The affinity of RSCt for DNA is reduced in the presence of AMP-PNP and ADP in a concentration-dependent manner with the addition of AMP-PNP having more pronounced effect. These differences in the magnitude at which the binding of ADP and AMP-PNP affects the affinity of DNA binding by RSCt suggest that the physical movement of the enzyme along DNA begins between the binding of ATP and its subsequent hydrolysis. Furthermore, the fact that the highest affinity for DNA binding by RSCt occurs in the absence of bound nucleotide offers a mechanistic explanation for the apparent low processivity of DNA translocation by the enzyme.

Substrate Binding Drives Large-Scale Conformational Changes in the Hsp90 Molecular Chaperone

Hsp90 is a ubiquitous molecular chaperone. Previous structural analysis demonstrated that Hsp90 can adopt a large number of structurally distinct conformations; however, the functional role of this flexibility is not understood. Here we investigate the structural consequences of substrate binding with a model system in which Hsp90 interacts with a partially folded protein (Δ131Δ), a well-studied fragment of staphylococcal nuclease. SAXS measurements reveal that under apo conditions, Hsp90 partially closes around Δ131Δ, and in the presence of AMPPNP, Δ131Δ binds with increased affinity to Hsp90's fully closed state. FRET measurements show that Δ131Δ accelerates the nucleotide-driven open/closed transition and stimulates ATP hydrolysis by Hsp90. NMR measurements reveal that Hsp90 binds to a specific, highly structured region of Δ131Δ. These results suggest that Hsp90 preferentially binds a locally structured region in a globally unfolded protein, and this binding drives functional changes in the chaperone by lowering a rate-limiting conformational barrier.

In Vivo Role of a Novel Kinesin-13 Microtubule Binding Site

Kinesin 13 family proteins regulate microtubule dynamics by using the energy of ATP hydrolysis to induce microtubule (MT) depolymerization. They have important roles in regulating MT dynamics during mitosis. Previous work in our lab identified a novel site of interaction of kinesin13 with the MT that leads to the formation of rings wrapping the MT in the presence of AMP-PNP. Several positively charged residues at this site were shown to be required for the formation of the rings but not for depolymerization activity.

Acetylation of Alpha Tubulin Lysine-40 Alone is not Sufficient for Changes in Kinesin-1 Motility

Kinesin-1 is a processive, plus end-directed motor that is responsible for major microtubule-based intracellular transport. It has been previously shown through in vivo studies that kinesin-1 preferentially translocates along certain subsets of microtubules, which are marked with specific posttranslational modifications (PTMs). We hypothesize that PTMs of tubulin directly influence the interaction of kinesin-1 with microtubules. In order to investigate the role of acetylation of α-tubulin at Lysine 40 (K40) in this context, we examined the binding affinity of kinesin in solution to acetylated and deacetylated microtubules in the presence of AMPPNP. We further characterized the single molecule motility properties of kinesin-1 on acetylated and deacetylated microtubules using Total Internal Reflection Fluorescence (TIRF) microscopy. To generate acetylated and deacetyalated microtubules, purified bovine tubulin was treated with the enzymes MEC-17 and SIRT2, respectively. Kinesin-1 motors were either expressed in COS cells or purified from bacterial cells. We found that kinesin-1 shows similar binding affinity, velocity and run length on acetylated and deacetylated microtubules as measured in these in vitro assays. Our results suggest that an alteration in the state of acetylation of K40 on α-tubulin in the microtubules does not result in changes in the catalytic cycle and strong or weak- binding states of the motor. We conclude that kinesin-1 cannot directly recognize the presence of an acetyl group on K40 of α-tubulin and hence this modification alone is not sufficient to explain the preferential motility of kinesin-1 observed in vivo. Rather, K40 acetylation appears to mark a subset of microtubules with other structural or biochemical alterations that are recognized as trafficking cues by kinesin-1.

A Two-step Mechanism for the Folding of Actin by the Yeast Cytosolic Chaperonin

Actin requires the chaperonin containing TCP1 (CCT), a hexadecameric ATPase essential for cell viability in eukaryotes, to fold to its native state. Following binding of unfolded actin to CCT, the cavity of the chaperone closes and actin is folded and released in an ATP-dependent folding cycle. In yeast, CCT forms a ternary complex with the phosducin-like protein PLP2p to fold actin, and together they can return nascent or chemically denatured actin to its native state in a pure in vitro folding assay. The complexity of the CCT-actin system makes the study of the actin folding mechanism technically challenging. We have established a novel spectroscopic assay through selectively labeling the C terminus of yeast actin with acrylodan and observe significant changes in the acrylodan fluorescence emission spectrum as actin is chemically unfolded and then refolded by the chaperonin. The variation in the polarity of the environment surrounding the fluorescent probe during the unfolding/folding processes has allowed us to monitor actin as it folds on CCT. The rate of actin folding at a range of temperatures and ATP concentrations has been determined for both wild type CCT and a mutant CCT, CCT4anc2, defective in folding actin in vivo. Binding of the non-hydrolysable ATP analog adenosine 5'-(β,γ-imino)triphosphate to the ternary complex leads to 3-fold faster release of actin from CCT following addition of ATP, suggesting a two-step folding process with a conformational change occurring upon closure of the cavity and a subsequent final folding step involving packing of the C terminus to the native-like state.

Decoration of microtubules in solution by the kinesin-14, Ncd.

The kinesin-14, Ncd, is a cellular motor involved in microtubule spindle assembly and contraction during mitosis and meiosis. Like other members of the kinesin superfamily, Ncd consists of two motor heads connected by a linker and a long cargo-carrying stalk. The motor heads hydrolyze ATP to ADP to provide the power stroke that moves them and the cargo along the microtubule. Whereas conventional kinesins move processively along the sense of the microtubule right-handed helix, Ncd moves in the opposite direction, apparently using a different motive mechanism. According to the current model, the microtubule-binding state of Ncd is bound by one head and then released during the motive cycle. This is distinguished from the binding states of conventional kinesins, in which the motor heads are always bound in the motive cycle with alternating one-head and two-head binding. The objective was to determine the extent of binding, the binding states of Ncd in the presence of an ATP analogue, AMPPNP, and whether the binding is cooperative. Small-angle neutron scattering (SANS) of microtubules decorated with a deuterated Ncd construct, Ncd281, in solution containing 42% D(2)O was used. These conditions render the microtubule `invisible' to SANS, while amplifying the SANS from the Ncd constructs. In the presence of AMPPNP, 75% of Ncd281 was not bound. The remainder was bound cooperatively by one of its motor heads to the microtubule.

Mitotic Kinesin CENP-E Promotes Microtubule Plus-End Elongation

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis, including establishment of microtubule (MT)-chromosome linkage and movement of mono-oriented chromosomes on kinetochore microtubules for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus ends. A human CENP-E construct was engineered, expressed, and purified, and it yielded theCENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end-directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity-marked MTs exhibited CENP-E-promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 μm/30 min. Immunolocalization studies revealed that 80.8% of plus-end-elongated MTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongation in solution best fit a single exponential function (k(obs) = 5.1 s(-1)), which is indicative of a mechanism in which α,β-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E, as part of its function in chromosome kinetochore-MT linkage, plays a direct role in MT elongation.