Characterization of a KDM5 small molecule inhibitor with antiviral activity against hepatitis B virus.

Chronic hepatitis B (CHB) is a global health care challenge and a major cause of liver disease. To find new therapeutic avenues with a potential to functionally cure chronic Hepatitis B virus (HBV) infection, we performed a focused screen of epigenetic modifiers to identify potential inhibitors of replication or gene expression. From this work we identified isonicotinic acid inhibitors of the histone lysine demethylase 5 (KDM5) with potent anti-HBV activity. To enhance the cellular permeability and liver accumulation of the most potent KDM5 inhibitor identified (GS-080) an ester prodrug was developed (GS-5801) that resulted in improved bioavailability and liver exposure as well as an increased H3K4me3:H3 ratio on chromatin. GS-5801 treatment of HBV-infected primary human hepatocytes reduced the levels of HBV RNA, DNA and antigen. Evaluation of GS-5801 antiviral activity in a humanized mouse model of HBV infection, however, did not result in antiviral efficacy, despite achieving pharmacodynamic levels of H3K4me3:H3 predicted to be efficacious from the in vitro model. Here we discuss potential reasons for the disconnect between in vitro and in vivo efficacy, which highlight the translational difficulties of epigenetic targets for viral diseases.

Biochemical characterization of tirabrutinib and other irreversible inhibitors of Bruton's tyrosine kinase reveals differences in on - and off - target inhibition.

BACKGROUND: Bruton's tyrosine kinase (BTK) is a key component of the B-cell receptor (BCR) pathway and a clinically validated target for small molecule inhibitors such as ibrutinib in the treatment of B-cell malignancies. Tirabrutinib (GS-4059/ONO-4059) is a selective, once daily, oral BTK inhibitor with clinical activity against many relapsed/refractory B-cell malignancies. METHODS: Covalent binding of tirabrutinib to BTK Cys-481 was assessed by LC-MSMS analysis of BTK using compound as a variable modification search parameter. Inhibition potency of tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib against BTK and related kinases was studied in a dose-dependent manner either after a fixed incubation time (as used in conventional IC50 studies) or following a time course where inactivation kinetics were measured. RESULTS: Tirabrutinib irreversibly and covalently binds to BTK Cys-481. The inactivation efficiency kinact/Ki was measured and used to calculate selectivity among different kinases for each of the four inhibitors studied. Tirabrutinib showed a kinact/Ki value of 2.4 ± 0.6 × 104 M-1 s-1 for BTK with selectivity against important off-targets. CONCLUSIONS: For the BTK inhibitors tested in this study, analysis of the inactivation kinetics yielded a more accurate measurement of potency and selectivity than conventional single-time point inhibition measurements. Subtle but clear differences were identified between clinically tested BTK inhibitors which may translate into differentiated clinical efficacy and safety. GENERAL SIGNIFICANCE: This is the first study that offers a detailed side-by-side comparison of four clinically-relevant BTK inhibitors with respect to their inactivation of BTK and related kinases.

Protein Footprinting and X-ray Crystallography Reveal the Interaction of PD-L1 and a Macrocyclic Peptide.

Blocking interactions between PD-1 and PD-L1 opens a new era of cancer treatment involving immunity modulation. Although most immunotherapies use monoclonal antibodies, small-molecule inhibitors offer advantages. To facilitate development of small-molecule therapeutics, we implemented a rapid approach to characterize the binding interfaces of small-molecule inhibitors with PD-L1. We determined its interaction with a synthetic macrocyclic peptide by using two mass spectrometry-based approaches, hydrogen-deuterium exchange and fast photochemical oxidation of proteins (FPOP), and corroborated the findings with our X-ray structure of the PD-L1/macrocycle complex. Although all three approaches show that the macrocycle binds directly to PD-L1 over the regions of residues 46-87 and 114-125, the two protein footprinting approaches show additional binding at the N-terminus of PD-L1, and FPOP reveals some critical binding residues. The outcomes not only show the binding regions but also demonstrate the utility of MS-based footprinting in probing protein/ligand inhibitory interactions in cancer immunotherapy.

The KN-93 Molecule Inhibits Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) Activity by Binding to Ca2+/CaM.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93-based mechanism-of-action studies and drug discovery efforts.

GS-5806 inhibits pre- to postfusion conformational changes of the respiratory syncytial virus fusion protein.

GS-5806 is a small-molecule inhibitor of human respiratory syncytial virus fusion protein-mediated viral entry. During viral entry, the fusion protein undergoes major conformational changes, resulting in fusion of the viral envelope with the host cell membrane. This process is reproduced in vitro using a purified, truncated respiratory syncytial virus (RSV) fusion protein. GS-5806 blocked these conformational changes, suggesting a possible mechanism for antiviral activity.

Direct binding of ledipasvir to HCV NS5A: mechanism of resistance to an HCV antiviral agent.

Ledipasvir, a direct acting antiviral agent (DAA) targeting the Hepatitis C Virus NS5A protein, exhibits picomolar activity in replicon cells. While its mechanism of action is unclear, mutations that confer resistance to ledipasvir in HCV replicon cells are located in NS5A, suggesting that NS5A is the direct target of ledipasvir. To date co-precipitation and cross-linking experiments in replicon or NS5A transfected cells have not conclusively shown a direct, specific interaction between NS5A and ledipasvir. Using recombinant, full length NS5A, we show that ledipasvir binds directly, with high affinity and specificity, to NS5A. Ledipasvir binding to recombinant NS5A is saturable with a dissociation constant in the low nanomolar range. A mutant form of NS5A (Y93H) that confers resistance to ledipasvir shows diminished binding to ledipasvir. The current study shows that ledipasvir inhibits NS5A through direct binding and that resistance to ledipasvir is the result of a reduction in binding affinity to NS5A mutants.

Large-scale functional purification of recombinant HIV-1 capsid.

During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents.

Lipid-sensing high-throughput ApoA-I assays.

Apolipoprotein A-I (ApoA-I), a primary protein component of high-density lipoprotein (HDL), plays an important role in cholesterol metabolism mediating the formation of HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by adenosine triphosphate (ATP) binding cassette A1 (ABCA1). Insufficient ABCA1 activity may lead to increased risk of atherosclerosis due to reduced HDL formation and cholesterol efflux. The standard radioactive assay for measuring cholesterol transport to ApoA-I has low throughput and poor dynamic range, and it fails to measure phospholipid transfer. We describe the development of two sensitive, nonradioactive high-throughput assays that report on the lipidation of ApoA-I: a homogeneous assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) and a discontinuous assay that uses the label-free Epic platform. The TR-FRET assay employs HiLyte Fluor 647-labeled ApoA-I with N-terminal biotin bound to streptavidin-terbium. When fluorescent ApoA-I was incorporated into HDL, TR-FRET decreased proportionally to the increase in the ratio of lipids to ApoA-I, demonstrating that the assay was sensitive to the amount of lipid bound to ApoA-I. In the Epic assay, biotinylated ApoA-I was captured on a streptavidin-coated biosensor. Measured resonant wavelength shift was proportional to the amount of lipids associated with ApoA-I, indicating that the assay senses ApoA-I lipidation.

The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function.

Tegobuvir (TGV) is a novel non-nucleoside inhibitor (NNI) of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chemical activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further analysis reveals that the aberrantly migrating NS5B species contains the inhibitor molecule. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore analysis of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP- mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI) of the viral polymerase.

Three routes to suppression of the neurodegenerative phenotypes caused by kinesin heavy chain mutations.

Kinesin-1 is a motor protein that moves stepwise along microtubules by employing dimerized kinesin heavy chain (Khc) subunits that alternate cycles of microtubule binding, conformational change, and ATP hydrolysis. Mutations in the Drosophila Khc gene are known to cause distal paralysis and lethality preceded by the occurrence of dystrophic axon terminals, reduced axonal transport, organelle-filled axonal swellings, and impaired action potential propagation. Mutations in the equivalent human gene, Kif5A, result in similar problems that cause hereditary spastic paraplegia (HSP) and Charcot-Marie-Tooth type 2 (CMT2) distal neuropathies. By comparing the phenotypes and the complementation behaviors of a large set of Khc missense alleles, including one that is identical to a human Kif5A HSP allele, we identified three routes to suppression of Khc phenotypes: nutrient restriction, genetic background manipulation, and a remarkable intramolecular complementation between mutations known or likely to cause reciprocal changes in the rate of microtubule-stimulated ADP release by kinesin-1. Our results reveal the value of large-scale complementation analysis for gaining insight into protein structure-function relationships in vivo and point to possible paths for suppressing symptoms of HSP and related distal neuropathies.

Dithiothreitol causes HIV-1 integrase dimer dissociation while agents interacting with the integrase dimer interface promote dimer formation.

We have developed a homogeneous time-resolved fluorescence resonance energy transfer (FRET)-based assay that detects the formation of HIV-1 integrase (IN) dimers. The assay utilizes IN monomers that express two different epitope tags that are recognized by their respective antibodies, coupled to distinct fluorophores. Surprisingly, we found that dithiothreitol (DTT), a reducing agent essential for in vitro enzymatic activity of IN, weakened the interaction between IN monomers. This effect of DTT on IN is dependent on its thiol groups, since the related chemical threitol, which contains hydroxyls in place of thiols, had no effect on IN dimer formation. By studying mutants of IN, we determined that cysteines in IN appear to be dispensable for the dimer dissociation effect of DTT. Peptides derived from the IN binding domain (IBD) of lens epithelium derived growth factor/transcriptional coactivator p75 (LEDGF), a cellular cofactor that interacts with the IN dimer interface, were tested in this IN dimerization assay. These peptides, which compete with LEDGF for binding to IN, displayed an intriguing equilibrium binding dose-response curve characterized by a plateau rising to a peak, then descending to a second plateau. Mathematical modeling of this binding system revealed that these LEDGF-derived peptides promote IN dimerization and block subunit exchange between IN dimers. This dose-response behavior was also observed with a small molecule that interacts with the IN dimer interface and inhibits LEDGF binding to IN. In conclusion, this novel IN dimerization assay revealed that peptide and small molecule inhibitors of the IN-LEDGF interaction also stabilize IN dimers and promote their formation.

Visualizing ATP-dependent RNA translocation by the NS3 helicase from HCV.

The structural mechanism by which nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) translocates along RNA is currently unknown. HCV NS3 is an ATP-dependent motor protein essential for viral replication and a member of the superfamily 2 helicases. Crystallographic analysis using a labeled RNA oligonucleotide allowed us to unambiguously track the positional changes of RNA bound to full-length HCV NS3 during two discrete steps of the ATP hydrolytic cycle. The crystal structures of HCV NS3, NS3 bound to bromine-labeled RNA, and a tertiary complex of NS3 bound to labeled RNA and a non-hydrolyzable ATP analog provide a direct view of how large domain movements resulting from ATP binding and hydrolysis allow the enzyme to translocate along the phosphodiester backbone. While directional translocation of HCV NS3 by a single base pair per ATP hydrolyzed is observed, the 3' end of the RNA does not shift register with respect to a conserved tryptophan residue, supporting a "spring-loading" mechanism that leads to larger steps by the enzyme as it moves along a nucleic acid substrate.

The macroscopic rate of nucleic acid translocation by hepatitis C virus helicase NS3h is dependent on both sugar and base moieties.

The nonstructural protein 3 helicase (NS3h) of hepatitis C virus is a 3'-to-5' superfamily 2 RNA and DNA helicase that is essential for the replication of hepatitis C virus. We have examined the kinetic mechanism of the translocation of NS3h along single-stranded nucleic acid with bases uridylate (rU), deoxyuridylate (dU), and deoxythymidylate (dT), and have found that the macroscopic rate of translocation is dependent on both the base moiety and the sugar moiety of the nucleic acid, with approximate macroscopic translocation rates of 3 nt s(-1) (oligo(dT)), 35 nt s(-1) (oligo(dU)), and 42 nt s(-1) (oligo(rU)), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein for the respective substrates such that weaker affinity corresponded to faster translocation. The values of K(0.5) for NS3h translocation at a saturating ATP concentration are as follows: 3.3+/-0.4 microM nucleotide (poly(dT)), 27+/-2 microM nucleotide (poly(dU)), and 36+/-2 microM nucleotide (poly(rU)). Furthermore, results of the isothermal titration of NS3h with these oligonucleotides suggest that differences in TDeltaS(0) are the principal source of differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometries for NS3h translocation were identical for all three substrates (approximately 0.5 ATP molecule consumed per nucleotide translocated). This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates.

Crystal structures of HIV-1 reverse transcriptase with etravirine (TMC125) and rilpivirine (TMC278): implications for drug design.

Diarylpyrimidine (DAPY) non-nucleoside reverse transcriptase inhibitors (NNRTIs) have inherent flexibility, helping to maintain activity against a wide range of resistance mutations. Crystal structures were determined with wild-type and K103N HIV-1 reverse transcriptase with etravirine (TMC125) and rilpivirine (TMC278). These structures reveal a similar binding mode for TMC125 and TMC278, whether bound to wild-type or K103N RT. Comparison to previously published structures reveals differences in binding modes for TMC125 and differences in protein conformation for TMC278.

Visualizing the molecular interactions of a nucleotide analog, GS-9148, with HIV-1 reverse transcriptase-DNA complex.

GS-9148 ([5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl]-phosphonic acid) is a dAMP (2'-deoxyadenosine monophosphate) analog that maintains its antiviral activity against drug-resistant HIV. Crystal structures for HIV-1 reverse transcriptase (RT) bound to double-stranded DNA, ternary complexes with either GS-9148-diphosphate or 2'-deoxyadenosine triphosphate (dATP), and a post-incorporation structure with GS-9148 translocated to the priming site were obtained to gain insight into the mechanism of RT inhibition. The binding of either GS-9148-diphosphate or dATP to the binary RT-DNA complex resulted in the fingers subdomain closing around the incoming substrate. This produced up to a 9 A shift in the tips of the fingers subdomain as it closed toward the palm and thumb subdomains. GS-9148-diphosphate shows a similar binding mode as dATP in the nucleotide-binding site. Residues whose mutations confer resistance to nucleotide/nucleoside RT inhibitors, such as M184, Y115, L74, and K65, show little to no shift in orientation whether GS-9148-diphosphate or dATP is bound. One difference observed in binding is the position of the central ring. The dihydrofuran ring of GS-9148-diphosphate interacts with the aromatic side chain of Y115 more than does the ribose ring of dATP, possibly picking up a favorable pi-pi interaction. The ability of GS-9148-diphosphate to mimic the active-site contacts of dATP may explain its effective inhibition of RT and maintained activity against resistance mutations. Interestingly, the 2'-fluoro moiety of GS-9148-diphosphate was found in close proximity to the Q151 side chain, potentially explaining the observed moderately reduced susceptibly to GS-9148 conferred by Q151M mutation.

RNase H active site inhibitors of human immunodeficiency virus type 1 reverse transcriptase: design, biochemical activity, and structural information.

Pyrimidinol carboxylic acids were designed as inhibitors of HIV-1 RNase H function. These molecules can coordinate to two divalent metal ions in the RNase H active site. Inhibition of enzymatic activity was measured in a biochemical assay, but no antiviral effect was observed. Binding was demonstrated via a solid state structure of the isolated p15-Ec domain of HIV-1 RT showing inhibitor and two Mn(II) ions bound to the RNase H active site.

Phosphoethanolamine N-methyltransferase (PMT-1) catalyses the first reaction of a new pathway for phosphocholine biosynthesis in Caenorhabditis elegans.

The development of nematicides targeting parasitic nematodes of animals and plants requires the identification of biochemical targets not found in host organisms. Recent studies suggest that Caenorhabditis elegans synthesizes phosphocholine through the action of PEAMT (S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferases) that convert phosphoethanolamine into phosphocholine. Here, we examine the function of a PEAMT from C. elegans (gene: pmt-1; protein: PMT-1). Our analysis shows that PMT-1 only catalyses the conversion of phosphoethanolamine into phospho-monomethylethanolamine, which is the first step in the PEAMT pathway. This is in contrast with the multifunctional PEAMT from plants and Plasmodium that perform multiple methylations in the pathway using a single enzyme. Initial velocity and product inhibition studies indicate that PMT-1 uses a random sequential kinetic mechanism and is feedback inhibited by phosphocholine. To examine the effect of abrogating PMT-1 activity in C. elegans, RNAi (RNA interference) experiments demonstrate that pmt-1 is required for worm growth and development and validate PMT-1 as a potential target for inhibition. Moreover, providing pathway metabolites downstream of PMT-1 reverses the RNAi phenotype of pmt-1. Because PMT-1 is not found in mammals, is only distantly related to the plant PEAMT and is conserved in multiple parasitic nematodes of humans, animals and crop plants, inhibitors targeting it may prove valuable in human and veterinary medicine and agriculture.

Defining the role of phosphomethylethanolamine N-methyltransferase from Caenorhabditis elegans in phosphocholine biosynthesis by biochemical and kinetic analysis.

In plants and Plasmodium falciparum, the synthesis of phosphatidylcholine requires the conversion of phosphoethanolamine to phosphocholine by phosphoethanolamine methyltransferase (PEAMT). This pathway differs from the metabolic route of phosphatidylcholine synthesis used in mammals and, on the basis of bioinformatics, was postulated to function in the nematode Caenorhabditis elegans. Here we describe the cloning and biochemical characterization of a PEAMT from C. elegans (gene, pmt-2; protein, PMT-2). Although similar in size to the PEAMT from plants, which contain two tandem methyltransferase domains, PMT-2 retains only the C-terminal methyltransferase domain. RNA-mediated interference experiments in C. elegans show that PMT-2 is essential for worm viability and that choline supplementation rescues the RNAi-generated phenotype. Unlike the plant and Plasmodium PEAMT, which catalyze all three methylations in the pathway, PMT-2 catalyzes only the last two steps in the pathway, i.e., the methylation of phosphomonomethylethanolamine (P-MME) to phosphodimethylethanolamine (P-DME) and of P-DME to phosphocholine. Analysis of initial velocity patterns suggests a random sequential kinetic mechanism for PMT-2. Product inhibition by S-adenosylhomocysteine was competitive versus S-adenosylmethionine and noncompetitive versus P-DME, consistent with formation of a dead-end complex. Inhibition by phosphocholine was competitive versus each substrate. Fluorescence titrations show that all substrates and products bind to the free enzyme. The biochemical data are consistent with a random sequential kinetic mechanism for PMT-2. This work provides a kinetic basis for additional studies on the reaction mechanism of PEAMT. Our results indicate that nematodes also use the PEAMT pathway for phosphatidylcholine biosynthesis. If the essential role of PMT-2 in C. elegans is conserved in parasitic nematodes of mammals and plants, then inhibition of the PEAMT pathway may be a viable approach for targeting these parasites with compounds of medicinal or agronomic value.

Autoinhibition of Escherichia coli Rep monomer helicase activity by its 2B subdomain.

DNA helicases catalyze separation of double-helical DNA into its complementary single strands, a process essential for DNA replication, recombination, and repair. The Escherichia coli Rep protein, a superfamily 1 DNA helicase, functions in DNA replication restart and is required for replication of several bacteriophages. Monomers of Rep do not display helicase activity in vitro; in fact, DNA unwinding requires Rep dimerization. Here we show that removal of the 2B subdomain of Rep to form RepDelta2B activates monomer helicase activity, albeit with limited processivity. Although both full length Rep and RepDelta2B monomers can translocate with 3' to 5' directionality along single-stranded DNA, the 2B subdomain inhibits the helicase activity of full length Rep. This suggests an autoregulatory mechanism for Rep helicase, which may apply to other nonhexameric helicases, whereby helicase activity is regulated by the rotational conformational state of the 2B subdomain; formation of a Rep dimer may relieve autoinhibition by altering the 2B subdomain orientation.

Microtubule-kinesin interface mutants reveal a site critical for communication.

Strict coordination of the two motor domains of kinesin is required for driving the processive movement of organelles along microtubules. Glutamate 164 of the kinesin heavy chain was shown to be critical for kinesin function through in vivo genetics in Drosophila melanogaster. The mutant motor E164K exhibited reduced steady-state ATPase activity and higher affinity for both ATP and microtubules. Moreover, an alanine substitution at this position (E164A) caused similar defects. It became stalled on the microtubule and was unable to bind and hydrolyze ATP at the second motor domain. Glu(164), which has been conserved through evolution, is located at the motor-microtubule interface close to key residues on helix alpha12 of beta-tubulin. We explored further the contributions of Glu(164) to motor function using several site-directed mutant proteins: E164K, E164N, E164D, E164Q, and D165A. The results indicate that the microtubule-E164K complex can only bind and hydrolyze one ATP. ATP with increased salt was able to dissociate a population of E164K motors from the microtubule but could not dissociate E164A. We tested the basis of the stabilized microtubule interaction with E164K, E164N, and E164A. The results provide new insights about the motor-microtubule interface and the pathway of communication for processive motility.