Kinesin-2 motors: Kinetics and biophysics.

Kinesin-2s are major transporters of cellular cargoes. This subfamily contains both homodimeric kinesins whose catalytic domains result from the same gene product and heterodimeric kinesins with motor domains derived from two different gene products. In this Minireview, we focus on the progress to define the biochemical and biophysical properties of the kinesin-2 family members. Our understanding of their mechanochemical capabilities has been advanced by the ability to identify the kinesin-2 genes in multiple species, expression and purification of these motors for single-molecule and ensemble assays, and development of new technologies enabling quantitative measurements of kinesin activity with greater sensitivity.

Skip residues modulate the structural properties of the myosin rod and guide thick filament assembly.

The rod of sarcomeric myosins directs thick filament assembly and is characterized by the insertion of four skip residues that introduce discontinuities in the coiled-coil heptad repeats. We report here that the regions surrounding the first three skip residues share high structural similarity despite their low sequence homology. Near each of these skip residues, the coiled-coil transitions to a nonclose-packed structure inducing local relaxation of the superhelical pitch. Moreover, molecular dynamics suggest that these distorted regions can assume different conformationally stable states. In contrast, the last skip residue region constitutes a true molecular hinge, providing C-terminal rod flexibility. Assembly of myosin with mutated skip residues in cardiomyocytes shows that the functional importance of each skip residue is associated with rod position and reveals the unique role of the molecular hinge in promoting myosin antiparallel packing. By defining the biophysical properties of the rod, the structures and molecular dynamic calculations presented here provide insight into thick filament formation, and highlight the structural differences occurring between the coiled-coils of myosin and the stereotypical tropomyosin. In addition to extending our knowledge into the conformational and biological properties of coiled-coil discontinuities, the molecular characterization of the four myosin skip residues also provides a guide to modeling the effects of rod mutations causing cardiac and skeletal myopathies.

Homodimeric Kinesin-2 KIF3CC Promotes Microtubule Dynamics.

KIF3C is one subunit of the functional microtubule-based kinesin-2 KIF3AC motor, an anterograde cargo transporter in neurons. However, KIF3C has also been implicated as an injury-specific kinesin that is a key regulator of axonal growth and regeneration by promoting microtubule dynamics for reorganization at the neuronal growth cone. To test its potential role as a modulator of microtubule dynamics in vitro, an engineered homodimeric KIF3CC was incorporated into a dynamic microtubule assay and examined by total internal reflection fluorescence microscopy. The results reveal that KIF3CC is targeted to the microtubule plus-end, acts as a potent catastrophe factor through an increase in microtubule catastrophe frequency, and does so by elimination of the dependence of the catastrophe rate on microtubule lifetime. Moreover, KIF3CC accelerates the catastrophe rate without altering the microtubule growth rate. Therefore, the ATP-promoted KIF3CC mechanism of catastrophe is different from the well-described catastrophe factors kinesin-13 MCAK and kinesin-8 Kip3/KIF18A. The properties of KIF3CC were not shared by heterodimeric KIF3AC and required the unique KIF3C-specific sequence extension in loop L11 at the microtubule interface. At the microtubule plus-end, the presence of KIF3CC resulted in modulation of the tapered structure typically seen in growing dynamic microtubules to microtubule blunt plus-ends. Overall our results implicate homodimeric KIF3CC as a unique promoter of microtubule catastrophe and substantiate its physiological role in cytoskeletal remodeling.

Determinants and Expansion of Specificity in a Trichothecene UDP-Glucosyltransferase from Oryza sativa.

Family 1 UDP-glycosyltransferases (UGTs) in plants primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, which is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin, and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins because of its ability to glycosylate DON, nivalenol, and hydrolyzed T-2 toxin (HT-2). However, Os79 is unable to modify T-2 toxin (T-2), produced by pathogens such as Fusarium sporotrichioides and Fusarium langsethii. Activity toward T-2 is desirable because it would allow a single UGT to inactivate co-occurring mycotoxins. Here, the structure of Os79 in complex with the products UDP and deoxynivalenol 3-O-glucoside is reported together with a kinetic analysis of a broad range of trichothecene mycotoxins. Residues associated with the trichothecene binding pocket were examined by site-directed mutagenesis that revealed that trichothecenes substituted at the C4 position, which are not glycosylated by wild-type Os79, can be accommodated in the binding pocket by increasing its volume. The H122A/L123A/Q202L triple mutation, which increases the volume of the active site and attenuates polar contacts, led to strong and equivalent activity toward trichothecenes with C4 acetyl groups. This mutant enzyme provides the broad specificity required to control multiple toxins produced by different Fusarium species and chemotypes.

Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod.

X-ray structural determination of segments of the myosin rod has proved difficult because of the strong salt-dependent aggregation properties and repeating pattern of charges on the surface of the coiled-coil that lead to the formation of paracrystals. This problem has been resolved in part through the use of globular assembly domains that improve protein folding and prevent aggregation. The primary consideration now in designing coiled-coil fusion constructs for myosin is deciding where to truncate the coiled-coil and which amino acid residues to include from the folding domain. This is especially important for myosin that contains numerous regions of low predicted coiled-coil propensity. Here we describe the strategy adopted to determine the structure of the region that extends from Arg1677 - Leu1797 that included two areas that do not show a strong sequence signature of a conventional left-handed coiled coil or canonical heptad repeat. This demonstrates again that, with careful choice of fusion constructs, overlapping structures exhibit very similar conformations for the myosin rod fragments in the canonical regions. However, conformational variability is seen around Leu1706 which is a hot spot for cardiomyopathy mutations suggesting that this might be important for function.

Reversible swelling of SBMV is associated with reversible disordering.

The structures of the compact and swollen southern bean mosaic virus (SBMV) particles have been compared by X-ray diffraction and proton magnetic resonance (PMR). Small-angle X-ray scattering showed that removal of divalent cations at alkaline pH causes the particle diameter to increase from 289Å in the native SBMV by 12% in solution and by 9% in microcrystals. The swelling is fully reversible upon re-addition of Ca2+ and Mg2+ ions, as shown by the X-ray patterns at 6Å resolution and by the 270MHz PMR spectra. Beyond 30Å resolution, X-ray patterns from the compact SBMV in solution and in microcrystals show fine fringes of ∼1/225Å-1 width extending to 6Å resolution, whereas patterns from the swollen SBMV in solution and in microcrystals show only broader fringes of ∼1/90Å-1 width, Model calculations demonstrate that the fine fringes from compact SBMV arise from regular packing of the protein subunits on the icosahedral surface lattice; the smearing of fine fringes in the swollen virus pattern can be simulated by uncorrelated displacements of pentamers and hexamers of protein subunits, with a standard deviation of 6Å from their mean locations. The PMR spectrum of compact SBMV is poorly resolved, whereas PMR spectrum of swollen SBMV shows sharp resonances in the methyl proton region. The line-narrowing for a fraction of the aliphatic protons upon swelling cannot be accounted for by rotational relaxation of the particle of 6×106MW, but must be attributed to internal motion in small regions of the protein subunits.

Fast or Slow, Either Head Can Start the Processive Run of Kinesin-2 KIF3AC.

Mammalian KIF3AC contains two distinct motor polypeptides and is best known for its role in organelle transport in neurons. Our recent studies showed that KIF3AC is as processive as conventional kinesin-1, suggesting that their ATPase mechanochemistry may be similar. However, the presence of two different motor polypeptides in KIF3AC implies that there must be a cellular advantage for the KIF3AC heterodimer. The hypothesis tested was whether there is an intrinsic bias within KIF3AC such that either KIF3A or KIF3C initiates the processive run. To pursue these experiments, a mechanistic approach was used to compare the pre-steady-state kinetics of KIF3AC to the kinetics of homodimeric KIF3AA and KIF3CC. The results indicate that microtubule collision at 11.4 µM(-1) s(-1) coupled with ADP release at 78 s(-1) are fast steps for homodimeric KIF3AA. In contrast, KIF3CC exhibits much slower microtubule association at 2.1 µM(-1) s(-1) and ADP release at 8 s(-1). For KIF3AC, microtubule association at 6.6 µM(-1) s(-1) and ADP release at 51 s(-1) are intermediate between the constants for KIF3AA and KIF3CC. These results indicate that either KIF3A or KIF3C can initiate the processive run. Surprisingly, the kinetics of the initial event of microtubule collision followed by ADP release for KIF3AC is not equivalent to 1:1 mixtures of KIF3AA plus KIF3CC homodimers at the same motor concentration. These results reveal that the intermolecular communication within the KIF3AC heterodimer modulates entry into the processive run regardless of whether the run is initiated by the KIF3A or KIF3C motor domain.

Family-specific Kinesin Structures Reveal Neck-linker Length Based on Initiation of the Coiled-coil.

Kinesin-1, -2, -5, and -7 generate processive hand-over-hand 8-nm steps to transport intracellular cargoes toward the microtubule plus end. This processive motility requires gating mechanisms to coordinate the mechanochemical cycles of the two motor heads to sustain the processive run. A key structural element believed to regulate the degree of processivity is the neck-linker, a short peptide of 12-18 residues, which connects the motor domain to its coiled-coil stalk. Although a shorter neck-linker has been correlated with longer run lengths, the structural data to support this hypothesis have been lacking. To test this hypothesis, seven kinesin structures were determined by x-ray crystallography. Each included the neck-linker motif, followed by helix α7 that constitutes the start of the coiled-coil stalk. In the majority of the structures, the neck-linker length differed from predictions because helix α7, which initiates the coiled-coil, started earlier in the sequence than predicted. A further examination of structures in the Protein Data Bank reveals that there is a great disparity between the predicted and observed starting residues. This suggests that an accurate prediction of the start of a coiled-coil is currently difficult to achieve. These results are significant because they now exclude simple comparisons between members of the kinesin superfamily and add a further layer of complexity when interpreting the results of mutagenesis or protein fusion. They also re-emphasize the need to consider factors beyond the kinesin neck-linker motif when attempting to understand how inter-head communication is tuned to achieve the degree of processivity required for cellular function.

Heterodimerization of Kinesin-2 KIF3AB Modulates Entry into the Processive Run.

Mammalian KIF3AB is an N-terminal processive kinesin-2 that is best known for its roles in intracellular transport. There has been significant interest in KIF3AB to define the key principles that underlie its processivity but also to define the mechanistic basis of its sensitivity to force. In this study, the kinetics for entry into the processive run were quantified. The results show for KIF3AB that the kinetics of microtubule association at 7 µm-1 s-1 is less than the rates observed for KIF3AA at 13 µm-1 s-1 or KIF3BB at 11.9 µm-1 s-1 ADP release after microtubule association for KIF3AB is 33 s-1 and is significantly slower than ADP release from homodimeric KIF3AA and KIF3BB, which reach 80-90 s-1 To explore the interhead communication implied by the rate differences at these first steps, we compared the kinetics of KIF3AB microtubule association followed by ADP release with the kinetics for mixtures of KIF3AA plus KIF3BB. Surprisingly, the kinetics of KIF3AB are not equivalent to any of the mixtures of KIF3AA + KIF3BB. In fact, the transients for each of the mixtures overlay the transients for KIF3AA and KIF3BB. These results reveal that intermolecular communication within the KIF3AB heterodimer modulates entry into the processive run, and the results suggest that it is the high rate of microtubule association that drives rebinding to the microtubule after force-dependent motor detachment.

Functional characterization of a soluble NADPH-cytochrome P450 reductase from Fusarium graminearum.

Fusarium head blight is a devastating disease in wheat caused by some fungal pathogens of the Fusarium genus mainly F. graminearum, due to accumulation of toxic trichothecenes. Most of the trichothecene biosynthetic pathway has been mapped, although some proteins of the pathway remain uncharacterized, including an NADPH-cytochrome P450 reductase. We subcloned a F. graminearum cytochrome P450 reductase that might be involved in the trichothecene biosynthesis. It was expressed heterologously in E. coli as N-terminal truncated form with an octahistidine tag for purification. The construct yielded a soluble apoprotein and its incubation with flavins yielded the corresponding monomeric holoprotein. It was characterized for activity in the pH range 5.5-9.5, using thiazolyl blue tetrazolium bromide (MTT) or cytochrome c as substrates. Binding of the small molecule MTT was weaker than for cytochrome c, however, the rate of MTT reduction was faster. Contrary to other studies of cytochrome reductase proteins, MTT reduction proceeded in a cooperative manner in our studies. Optimum kinetic activity was found at pH 7.5-8.5 for bothMTT and cytochrome c. This is the first paper presenting characterization of a cytochrome P450 reductase from F. graminearum which most likely is involved in mycotoxin biosynthesis or some primary metabolic pathway such as sterol biosynthesis in F. graminearum.

Crystal Structure of Os79 (Os04g0206600) from Oryza sativa: A UDP-glucosyltransferase Involved in the Detoxification of Deoxynivalenol.

Fusarium head blight is a plant disease with significant agricultural and health impact which affects cereal crops such as wheat, barley, and maize and is characterized by reduced grain yield and the accumulation of trichothecene mycotoxins such as deoxynivalenol (DON). Studies have identified trichothecene production as a virulence factor in Fusarium graminearum and have linked DON resistance to the ability to form DON-3-O-glucoside in wheat. Here, the structures of a deoxynivalenol:UDP-glucosyltransferase (Os79) from Oryza sativa are reported in complex with UDP in an open conformation, in complex with UDP in a closed conformation, and in complex with UDP-2-fluoro-2-deoxy-d-glucose and trichothecene at 1.8, 2.3, and 2.2 Å resolution, respectively. The active site of Os79 lies in a groove between the N-terminal acceptor and the C-terminal donor-binding domains. Structural alignments reveal that Os79 likely utilizes a catalytic mechanism similar to those of other plant UGTs, with His 27 activating the trichothecene O3 hydroxyl for nucleophilic attack at C1' of the UDP-glucose donor. Kinetic analysis of mutant Os79 revealed that Thr 291 plays a critical role in catalysis as a catalytic acid or to position the UDP moiety during the nucleophilic attack. Steady-state kinetic analysis demonstrated that Os79 conjugates multiple trichothecene substrates such as DON, nivalenol, isotrichodermol, and HT-2 toxin, but not T-2 toxin. These data establish a foundation for understanding substrate specificity and activity in this enzyme and can be used to guide future efforts to increase DON resistance in cereal crops.

A composite approach towards a complete model of the myosin rod.

Sarcomeric myosins have the remarkable ability to form regular bipolar thick filaments that, together with actin thin filaments, constitute the fundamental contractile unit of skeletal and cardiac muscle. This has been established for over 50 years and yet a molecular model for the thick filament has not been attained. In part this is due to the lack of a detailed molecular model for the coiled-coil that constitutes the myosin rod. The ability to self-assemble resides in the C-terminal section of myosin known as light meromyosin (LMM) which exhibits strong salt-dependent aggregation that has inhibited structural studies. Here we evaluate the feasibility of generating a complete model for the myosin rod by combining overlapping structures of five sections of coiled-coil covering 164 amino acid residues which constitute 20% of LMM. Each section contains ∼ 7-9 heptads of myosin. The problem of aggregation was overcome by incorporating the globular folding domains, Gp7 and Xrcc4 which enhance crystallization. The effect of these domains on the stability and conformation of the myosin rod was examined through biophysical studies and overlapping structures. In addition, a computational approach was developed to combine the sections into a contiguous model. The structures were aligned, trimmed to form a contiguous model, and simulated for >700 ns to remove the discontinuities and achieve an equilibrated conformation that represents the native state. This experimental and computational strategy lays the foundation for building a model for the entire myosin rod.

Kinesin-2 KIF3AC and KIF3AB Can Drive Long-Range Transport along Microtubules.

Mammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 µm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 µm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 µm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons.

Kinesin-2 KIF3AB exhibits novel ATPase characteristics.

KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. There has been significant interest in KIF3AB in defining the key principles that underlie the processivity of KIF3AB in comparison with homodimeric processive kinesins. To define the ATPase mechanism and coordination of KIF3A and KIF3B stepping, a presteady-state kinetic analysis was pursued. For these studies, a truncated murine KIF3AB was generated. The results presented show that microtubule association was fast at 5.7 µm(-1) s(-1), followed by rate-limiting ADP release at 12.8 s(-1). ATP binding at 7.5 µm(-1) s(-1) was followed by an ATP-promoted isomerization at 84 s(-1) to form the intermediate poised for ATP hydrolysis, which then occurred at 33 s(-1). ATP hydrolysis was required for dissociation of the microtubule·KIF3AB complex, which was observed at 22 s(-1). The dissociation step showed an apparent affinity for ATP that was very weak (K½,ATP at 133 µm). Moreover, the linear fit of the initial ATP concentration dependence of the dissociation kinetics revealed an apparent second-order rate constant at 0.09 µm(-1) s(-1), which is inconsistent with fast ATP binding at 7.5 µm(-1) s(-1) and a Kd ,ATP at 6.1 µm. These results suggest that ATP binding per se cannot account for the apparent weak K½,ATP at 133 µm. The steady-state ATPase Km ,ATP, as well as the dissociation kinetics, reveal an unusual property of KIF3AB that is not yet well understood and also suggests that the mechanochemistry of KIF3AB is tuned somewhat differently from homodimeric processive kinesins.

Insights into the specificity of lysine acetyltransferases.

Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. Here we report the structure of a GNAT in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.

Dissecting cobamide diversity through structural and functional analyses of the base-activating CobT enzyme of Salmonella enterica.

BACKGROUND: Cobamide diversity arises from the nature of the nucleotide base. Nicotinate mononucleotide (NaMN):base phosphoribosyltransferases (CobT) synthesize α-linked riboside monophosphates from diverse nucleotide base substrates (e.g., benzimidazoles, purines, phenolics) that are incorporated into cobamides. METHODS: Structural investigations of two members of the CobT family of enzymes in complex with various substrate bases as well as in vivo and vitro activity analyses of enzyme variants were performed to elucidate the roles of key amino acid residues important for substrate recognition. RESULTS: Results of in vitro and in vivo studies of active-site variants of the Salmonella enterica CobT (SeCobT) enzyme suggest that a catalytic base may not be required for catalysis. This idea is supported by the analyses of crystal structures that show that two glutamate residues function primarily to maintain an active conformation of the enzyme. In light of these findings, we propose that proper positioning of the substrates in the active site triggers the attack at the C1 ribose of NaMN. CONCLUSION: Whether or not a catalytic base is needed for function is discussed within the framework of the in vitro analysis of the enzyme activity. Additionally, structure-guided site-directed mutagenesis of SeCobT broadened its substrate specificity to include phenolic bases, revealing likely evolutionary changes needed to increase cobamide diversity, and further supporting the proposed mechanism for the phosphoribosylation of phenolic substrates. GENERAL SIGNIFICANCE: Results of this study uncover key residues in the CobT enzyme that contribute to the diversity of cobamides in nature.

Functional asymmetry in kinesin and dynein dimers.

Active transport along the microtubule lattice is a complex process that involves both the Kinesin and Dynein superfamily of motors. Transportation requires sophisticated regulation much of which occurs through the motor's tail domain. However, a significant portion of this regulation also occurs through structural changes that arise in the motor and the microtubule upon binding. The most obvious structural change being the manifestation of asymmetry. To a first approximation in solution, kinesin dimers exhibit twofold symmetry, and microtubules exhibit helical symmetry. The higher symmetries of both the kinesin dimers and microtubule lattice are lost on formation of the kinesin-microtubule complex. Loss of symmetry has functional consequences such as an asymmetric hand-over-hand mechanism in plus-end-directed kinesins, asymmetric microtubule binding in the Kinesin-14 family, spatially biased stepping in dynein and cooperative binding of additional motors to the microtubule. This review focusses on how the consequences of asymmetry affect regulation of motor heads within a dimer, dimers within an ensemble of motors, and suggests how these asymmetries may affect regulation of active transport within the cell.

Structural organization of FtsB, a transmembrane protein of the bacterial divisome.

We report the first structural analysis of an integral membrane protein of the bacterial divisome. FtsB is a single-pass membrane protein with a periplasmic coiled coil. Its heterologous association with its partner FtsL represents an essential event for the recruitment of the late components to the division site. Using a combination of mutagenesis, computational modeling, and X-ray crystallography, we determined that FtsB self-associates, and we investigated its structural organization. We found that the transmembrane domain of FtsB homo-oligomerizes through an evolutionarily conserved interaction interface where a polar residue (Gln 16) plays a critical role through the formation of an interhelical hydrogen bond. The crystal structure of the periplasmic domain, solved as a fusion with Gp7, shows that 30 juxta-membrane amino acids of FtsB form a canonical coiled coil. The presence of conserved Gly residue in the linker region suggests that flexibility between the transmembrane and coiled coil domains is functionally important. We hypothesize that the transmembrane helices of FtsB form a stable dimeric core for its association with FtsL into a higher-order oligomer and that FtsL is required to stabilize the periplasmic domain of FtsB, leading to the formation of a complex that is competent for binding to FtsQ, and to their consequent recruitment to the divisome. The study provides an experimentally validated structural model and identifies point mutations that disrupt association, thereby establishing important groundwork for the functional characterization of FtsB in vivo.

Common mechanistic themes for the powerstroke of kinesin-14 motors.

Kar3Cik1 is a heterodimeric kinesin-14 from Saccharomyces cerevisiae involved in spindle formation during mitosis and karyogamy in mating cells. Kar3 represents a canonical kinesin motor domain that interacts with microtubules under the control of ATP-hydrolysis. In vivo, the localization and function of Kar3 is differentially regulated by its interacting stoichiometrically with either Cik1 or Vik1, two closely related motor homology domains that lack the nucleotide-binding site. Indeed, Vik1 structurally resembles the core of a kinesin head. Despite being closely related, Kar3Cik1 and Kar3Vik1 are each responsible for a distinct set of functions in vivo and also display different biochemical behavior in vitro. To determine a structural basis for their distinct functional abilities, we used cryo-electron microscopy and helical reconstruction to investigate the 3-D structure of Kar3Cik1 complexed to microtubules in various nucleotide states and compared our 3-D data of Kar3Cik1 with that of Kar3Vik1 and the homodimeric kinesin-14 Ncd from Drosophila melanogaster. Due to the lack of an X-ray crystal structure of the Cik1 motor homology domain, we predicted the structure of this Cik1 domain based on sequence similarity to its relatives Vik1, Kar3 and Ncd. By molecular docking into our 3-D maps, we produced a detailed near-atomic model of Kar3Cik1 complexed to microtubules in two distinct nucleotide states, a nucleotide-free state and an ATP-bound state. Our data show that despite their functional differences, heterodimeric Kar3Cik1 and Kar3Vik1 and homodimeric Ncd, all share striking structural similarities at distinct nucleotide states indicating a common mechanistic theme within the kinesin-14 family.

Structural insights into the substrate specificity of the Rhodopseudomonas palustris protein acetyltransferase RpPat: identification of a loop critical for recognition by RpPat.

Lysine acetylation is a post-translational modification that is important for the regulation of metabolism in both prokaryotes and eukaryotes. In bacteria, the best studied protein acetyltransferase is Pat. In the purple photosynthetic bacterium Rhodopseudomonas palustris, at least 10 AMP-forming acyl-CoA synthetase enzymes are acetylated by the Pat homologue RpPat. All bona fide RpPat substrates contain the conserved motif PX(4)GK. Here, we show that the presence of such a motif is necessary but not sufficient for recognition by RpPat. RpPat failed to acetylate the methylmalonyl-CoA synthetase of this bacterium (hereafter RpMatB) in vivo and in vitro, despite the homology of RpMatB to known RpPat substrates. We used RpMatB to identify structural determinants that are recognized by RpPat. To do this, we constructed a series of RpMatB chimeras that became substrates of RpPat. In such chimeras, a short region (11-25 residues) of RpMatB located >20 residues N-terminal to the acetylation site was replaced with the corresponding sequences from other AMP-forming acyl-CoA synthetases that were known RpPat substrates. Strikingly, the enzymatic activity of RpMatB chimeras was regulated by acetylation both in vitro and in vivo. Crystal structures of two of these chimeras showed that the major difference between them and wild-type RpMatB was within a loop region ∼23 Šfrom the acetylation site. On the basis of these results, we suggest that RpPat likely interacts with a relatively large surface of its substrates, in addition to the PX(4)GK motif, and that RpPat probably has relatively narrow substrate specificity.