Nano-manipulation of actomyosin molecular motors in vitro: a new working principle

TIBS 1 8 - SEPTEMBER 1993

MUSCLECONTRACTIONand other biological motions are propelled by an actomyosin motor driven by the chemical energy of ATP hydrolysis. Despite numerous studies, the working principle of this molecular motor remains elusive. The underlying mechanism is not as simple as that expected from analogy with man-made machines. Since a molecular motor is only nanometers in size and has a flexible structure, it is very prone to thermal agitation. Molecular motors can thus operate under the strong influence of this thermal noise, with a high efficiency of chemo-mechanical energy conversion (40% at maximum; Ref. I). This is in sharp contrast to man-made machines which operate at energies much higher than the thermal noise. To elucidate the working principle of the molecular motor, it is essential to resolve the intrinsic characteristics of the molecular machine2. Until recently, the mechanism of motion underlying molecular motors was studied using muscle fibers, or suspensions of purlfied motor proteins. These systems, however, are too complex to provide unambiguous information about the elementary process of energy transduction by individual molecular motors. To alleviate this problem, new techniques were needed that could directly probe the elementary process at the molecular level. Several kinds of in vitro motility models using purified motor proteins have now been designed (see Ref. 3 for a review). A model that allows the direct observation of single actin filaments labeled with fluorescent phalloidin by optical microscopy4, as well as the motion produced by myosin or its subfragments bound to a substrate s,6, has received a great deal of attention. Furthermore, a very subtle technique for manipulating a single actin filament by a fine glass needle has been developed~, which has allowed the measurement of both the motion and force of individual motors

Nano-manipulation of acto'my: sin

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molecular motors in vitro: a,,new *

"working principle Toshio Yanagida, Yoshie Harada and Akihiko Ishijima Techniques have been recently developed that allow the direct observation of single actin filaments and their manipulation, using glass microneedles, in the nanometer range. Further development of these techniques has made possible the detection of subpiconewton-level forces of individual myosin heads. This in vitro motility model is sensitive in the submillisecond range and has allowed us to determine the force generation of an actomyosin motor directly at the molecular level. The results have led to a new conceptual framework for chemo-mechanical energy transduction in the molecular motor.

in vitro at very high resolution (subnanometer and subpiconewton, respectively; Ref. 8). These new in vitro motility assays have greatly enhanced the progress of research on the actomyosin molecular motor. In this review, we will survey high-resolution measurements of both the motion and force of the molecular motor and introduce a new conceptual framework for chemomechanical energy transduction.

Direct observation of motor proteins and an In vitro motility model

The first step when studying the elementary process of chemo-mechanical energy transduction is to directly observe the motion of a molecular motor in solution by optical microscopy. The resolution of conventional optical microscopes (-0.2 pin), however, is too low to observe single protein molecules, or even a small assembly of motors. Thus a great deal of effort has been made to increase the resolution of the experimental system. One method is to observe the scattered light from objects using a dark-field microscope equipped with a very strong illuminating source and a highly sensitive T. ¥ana~da is at the Departmentof camera. This method has the advantage BiophysicalEngineering,OsakaUniversity, that objects can be observed without Toyonaka,Osaka,Japan.¥. Haradaand any type of labeling. However, the obA. IshlJimaare at the ERATOBIO-MOTRON jects observed need to be as large as a Project, Senba-Higashi2414, Mino,Osaka, microtubule, about 20 nm in diameter9. A Japan. © 1993,ElsevierSciencePublishers,(UK)0968-0004/93/$06.00

similar resolution can be achieved if the contrast of the recording is greatly enhanced by computer image processingI° (video-enhanced contrast method). Fluorescence microscopy is useful when observing smaller objects n. The number of photons emitted from a single molecule of fluorescent dye can be observed by a fluorescence microscope equipped with conventional highsensitivity detectors, such as a silicon intensified target (SIT) camera or an image intensifier 12, It is theoretically possible to observe single protein molecules labeled with fluorescent dyes, although it has yet to be proven in practice owing to strong photobleaching effects. We have demonstrated that single actin filaments labeled with phalloidin-dye complexes (which stabilize the filament structure of actin but do not affect the ability to produce motion with myosin13) can be observed clearly and continuously by fluorescence microscopy4. This technique has allowed us to observe the motion of actin filaments during the interaction with myosin in the presence of ATP. Using this method, it has been shown that the bending motion of actin filaments is modulated by an interaction in solution with myosin subkagments (S-I and HMM) in the presence of ATP4. However, the unidirectional motion of actin filaments, which is evident in

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Box 1. Manipulationof motor proteinsby optical tweezers The single-beam optical gradient field trap (optical tweezers) is a To TV camera useful method to capture and manipulate small (25-100 pm) ~, Two-dimensional dielectric particles in solution2s. Optical tweezers have recently Laser beam I I positionsensor attracted much attention in the biologicalfield since with them cells High NA D M ~ p ~ = and subcellular components can be easily manipulated in a non~ ~ / j J objective rojection ~ -invasive manner without significant radiation damage28-3°. Furthermore, the technique is simple. The figure shows the optical tweezers ,~-,~ Bead DM/~ of bead system for manipulating a single actin filament attached to a latex bead, using a microscope equipped with epifluorescence optics. A laser beam is focused on the specimen by an objective with a high numerical aperture. The scattering force, pointing in the direction of the incident light, pushes the bead down and the gradient force pulls it into the region of high light intensity, i.e. the focal spot. The bead is thus trapped just below the focal spot where both of the forces balance each other. As the laser beam used has a gaussian intensity distribution in its cross-section, the bead is securely ~ ~ ' ~ mirrors trapped in the horizontal (X-Y) direction by the additional gradient force pulling it into the center of the beam. The trapping force DM : Dichroic mirror depends on the intensity of the laser and the size of the particle. Halogen lamp ~ With a I pm diameter latex bead and 100 mW laser power, the force produced is several tens of piconewtons, which is large enough to move the bead against the viscous drag in water at a velocity of 3700 pm s-1. The bead can be manipulated in two dimensions by moving the focal spot using the galvanometermirrors. As the actin filament is too small to be trapped, one end of the actin filament, labeledwith fluorescent phalloidin, is attached to the bead trapped in the movablebeam (1 pm in diameter). The bead is coated with NEM-treatedmyosin (see text) and fluorescently labeled so the actin filament and the bead can be observed simultaneouslyunder a fluorescence microscope. Thus, it is possible to manipulate actin filaments. When the other end of the actin filament is brought into contact with the myosin-coatedsurface, the force due to actomyosin interaction can be measured. Sincethe light intensity distribution of the laser beam is gaussian, the trapping force increases proportionallywith the deviation of the position of the center of the bead from the center of the laser beam. Therefore,the force due to actomyosin interaction can be determined by measuringthis deviation. The deviation is measured by a two-dimensionalposition sensor with a space resolution in the nanometerrange by essentiallythe same method as that shown in the text, which correspondsto a force resolution in the piconewton range.

intact muscle, was not observed in solution. It has been shown that, when myosin or its subfragments were bound to the surface of a substrate, the actin moved In a unidirectional way along them with a velocity similar to that found in intact muscle5,~, Thus, it has been possible to observe the motion of molecular motors with the use of optical microscopy. This in vitro motility model has been widely used to test the functions of motor proteins, since it does not require any special skills or large-scale equipment. Mlcromanipulatlonof an actin filament and force measurementof the molecularmotor Observation of the motion of actin can only provide limited information about the mechanism of force generation. Recently, however, a technique for manipulating a single actin filament using a fine glass needle has been developed T. This technique enables one to exert a force on an actin filament, or to measure the force exerted on an actin filament when interacting with myosin. One end of an actin filament is caught by a glass micro-needle mounted on a mechanical manipulator; the other end is brought into contact with a glass sudace that has been coated with N-ethyl maleimide (NEM)-treated myosin in order to increase its affinity

32O

for actin. The actin filament is pulled by the force produced by the actomyosin interaction, bending the needle. The force is determined by measuring the degree of bending of the needle, as observed on a TV monitor. Using this method it is possible to measure a force of several plconewtons. Thus, the force as well as the motion of the actin filament has been measured using this in vitro motility model. Measurements of subnanometermotion and subplconewtonforce of the molecularmotor Another new technique that directly probes the process of the force generation of individual molecular motors in vitro has been developeds, In order to minimize the deterioration of the time resolution resulting from the needle's mass and viscous drag in the solution, a finer glass needle, 50-70 pm in length and 0.2 pm in diameter, has been used. This fine glass needle was attached to a rigid holding rod mounted on a piezoactuator and a mechanical manipulator that could move the needle over a range from less than 0.I nm to several tens of micrometers. The force was determined by measuring the displacement of the needle, as described above. In turn, the displacement was detected at very high time and space resolutions as follows.

The static position of the needle cannot be determined at a space resolution of less than about half of the wavelength of light owing to the diffraction limit. The displacement, however, is

different :~-:8. For instance, if the image of the needle is projected onto a pair of photo-dlodes and the displacement Is determined by measuring the difference in the photo-currents of the photodiodes, a displacement of less than I/I000 of the wavelength can be detected. This method is similar to that used in an atomic force microscope (AFM) to determine the position of the probe. The function of the pair of photodiodes can be replaced by computer processing images of the needles, or small particles attached to objects, which have been recorded on video tape. The time resolution, however, is not as high 19 (several tens of milliseconds). The bending of the needle is measured by the system shown in Fig. I. To increase the contrast, a small nickel particle of about 1 Bm in diameter is attached to the tip of the needle. An image of the particle is projected onto a pair of photo-diodes, and the displacement of the needle is obtained from the differential output of the photodetector. The differential output is very sensitive to any movement of the particle

TIBS 18 - SEPTEMBER 1993

(as mentioned above) and is linear for motions of 0.1-100 nm. The lower limit of the movement detection is less than 0.I nm. Since the stiffness of the needles used is in the range of 0.01-I0 pN nm-t, a displacement of 0.I nm corresponds to forces of 0.001-I.0 pN. The rise time of the tip of the needle, when the base of the needle is suddenly moved by the piezoactuator, is less than 0.2 ms. Similar measurements are possible using optical tweezers (Box I). It is worth noting that, although the system can d~tect the displacement of the needle with high space and time resolutions, the accuracy of the force and movement measurements produced by actomyosin motors is limited by thermal fluctuations of the needle. The root mean square (rms) error in a force measurement based on a single recording is given as ~/error ~/KkBT, where K is the stiffness of the needle, ka the Boltzmann constant and T the absolute temperature 2°. For instance, when K= 1 and 0.01 pN nm-I, ~J
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Rgure 1 Micromanipulation of a single actin filament by a glass microneedle and subpiconewton force measurement system in vitros. Single actin filaments, labeled with fluorescent phalloidin, were observed using an invertedfluorescence microscope. The images were videotapedby a high sensitivity (SIT) TV camera and a video recorder, and projected onto a TV monitor. By using the monitor, one end of an actin filament was caught by a glass microneedlethat was mounted on a mechanical manipulator and a piezo-actuatorwhile the other end was brought into contact with the myosin-coatedglass surface in the presenceof ATP. The force was determined by measuringthe displacementof the needle resulting from the actomyosin interaction. The image of the needle was projected onto a pair of photodiodes, and the displacementof the needle was determinedfrom the differential photocurrants. Thus, the displacement could be measured at resolutions of 0.1 nm, corresponding to ~ 0.1 pN and 0.2 ms, respectively.

to achieve a greater accuracy of simultaneous force and displacement measurements based on a single recording. This limitation (4pN nm), however, is smaller than the size of a single power stroke produced by a single myosin head under near isometric conditions, in which force and displacement are expected to be approximately 2 pN and I0 nm, respectively. This does not apply when we reduce the thermal rise by filtering it out or when we deal with the steady-state process, and data

can be recorded for a long period of time (for instance, force fluctuations during isometric conditions or sliding as shown below). In such cases, it is possv ble to achieve much greater accuracy.

Force produced by individual molecular motors Figure 2a shows a typical time course of force generated by minimum sliding,

obtained by measuring the needle displacements caused by a small number of myosin heads interacting with an actin filament, i.e. at near isometric conditions. The force fluctuates greatly, since the number of myosin heads involved in the force generation is very small (approximately five, based on a noise analysis). The fluctuations observed before the arrow are due to the

321

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thermal vibrations of the free needle. undergo attachment force generation pendently, and one power stroke correThe noise analysis of the force fluctu- (power stroke.) and detachment cycles sponds to each ATPase cycle (Fig. 3a). ations showed that the myosin heads with actin both stochastically and inde- Therefore, some of the force impulses in Fig. 2a should be those produced by individual heads; thus a mechanical event produced by a single molecule, coupled to the ATPase reaction, has been de(a) tected for the first time. More direct recording of a single-motor force, comparable to recording a single-channel current in a membrane, is currently in progress. When the number of myosin heads was increased, the actin filament slid along the myosin-coated surface and the needle displacement was greater 2s (Fig. 2b, upper trace). The lower trace shows the motion of the needle on an expanded time scale during the sliding phase. Fluctuations of the needle position are visible as deviations from the smooth curve. These were obtained by subtracting the thermal vibrations of the free needle before the generation of force from the fluctuations during the sliding phase. Both are similar, indicatE c ing that the force fluctuations during sliding are very small. The amplitude of z the response is 1/6 to 1/10 of that near o isometric conditions when the same number of myosin heads are involved. In this example, the average velocity is about 2 Ixms4, which corresponds to 25% of the maximum velocity at zero load. At greater than I l~ms-' similar results are obtained, and at less than p 0.2 pms -z the amplitude of the fluctu80 ~'~'7~' ations is as large as that under iso60 metric conditions. Thus, the amplitude E" 60 of the force fluctuations Is greatly dependent on either the velocity or the load. These results strongly Indicate 40 Q, E 40 that the actomyosin motor can modulate the coupling between the ATPase and the mechanical cycles, depending } 20 ,,o on the load.

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R~m 2 Force produced by individual myosin heads under (a) near isometric conditions and (b) during sliding. When the actin filaments were brought into contact with the myosin-coated surface at the arrows, the force developed immediately. The fluctuations before the force generations are the thermal vibrations of the free needles. On the basis of the random on-off kinetics model, the average force of a myosin head near isometric conditions was 0.42 pN (Ref. 8), Recently,the force obtained for a myosin head, correctly oriented on a myosin filamerit relative to the actin filament, was 2.3 pN (Ref. 26). In (b), the lower trace indicates the movement of the needle during the sliding phase on an expanded time scale. The insert indicates the fluctuations of the position of the needle (deviations from the smooth curve shown by the broken line). The average velocity during the sliding phase is 2 pms -1.

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A new wmkl~ principle for the aotomyosln molecular motor Until recently, the mechanism concerning the movement of the actomyosin molecular motor has been explained in terms of the crossbridge swingtng model 2z,22. ]n this model, the sliding force is due to attachment force generation (power stroke) and detachment cycles of actomyosin. This model assumes 'that the power stroke is pro-

duced by a large-scale structural change (a swinging motion) of the myosin head, and that one stroke cycle strictly corresponds to one ATPase cycle, independent of the load. The idea of a l : 1 tight coupling between chemical and

TIBS 18 - SEPTEMBER 1 9 9 3

mechanical reactions appears to result from the concept of allosteric proteins23. This idea has been generally assumed to explain the mechanisms of biological energy transduction. The force fluctuations near isometric conditions of a large load (Fig. 2a) are consistent with the 1 : 1 coupling (Fig. 3a). This idea, however, cannot be applied when the actin slides under a moderate load. As the sliding velocity increases, the period during which the myosin head can interact with an actin monomer decreases. Therefore, if the power stroke corresponds to a single ATPase cycle, as the velocity increases the power

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stroke time should become shorter and, consequently, p o+ the amplitude of force fluctuations would increase (Fig. 3b). The actual force fluctuations, however, Rgure 3 greatly decreased with New working principle of the actomyosin molecular motor. (a) Chemo-mechanical coupling under near velocity (>15% of the maxiisometric conditions. One power stroke corresponds to each ATPase cycle (1:1 coupling, left) and a mum velocity at zero load, myosin head produces an impulsive force (center), so that the force produced by multiple heads fluc1 pms -1, Fig. 2b), indicating tuates greatly (right). The observed force fluctuations are consistent with the 1:1 coupling near isometric conditions. (b), (e) Chemo-mechanical coupling during sliding under a moderate load. The conventhat the actomyosin motor tional model has assumed a 1:1 tight coupling (b). Since in this model one power stroke strictly produces an almost concorresponds to each ATPase cycle, independent of the load, a myosin head produces a sharper impulatant force during the sire force (center, see text) and the force fluctuations are larger (right). In a new model (©), the ATPase cycle8. The results chemo-mechanical coupling is variable, dependent on the load; each ATPase corresponds to one strongly indicate that actopower stroke with a large load, and multiple power strokes with a moderate load. A myosin head promyosin can perform multiduces an almost constant force during the ATPase cycle at a moderate load (center), and the force fluctuations are veff small (right), i.e. the actin is moved a long distance smoothly and efficiently. The ple power strokes during observed force fluctuations are consistent with the 1:many variable coupling model. one ATPase cycle during sliding, not only at zero, but also at moderate load. These molecule (-20kBT). Therefore, the the I:1 tight coupling hypothesis has results have important implications and actomyosin motor constitutes a very been widely accepted. In order to explain lead to the conclusion that the coupling skilful molecular machine that can the 1 : many variable coupling model, a between the ATPase and power stroke operate with very high efficiency= new conceptual framework is necescycles is variablr+; depending on the (>40% at maximum), even when the sary. Thus, an investigation of the actoinput energy level is close to the aver- myosin molecular motor is now enterload (Fig 3a, c). Since we first reported it24, the prob- age thermal energy (kBT). The mech- ing a new phase. Further development lem of whether the chemo-mechanical anism behind the gradual use of chemi- of this technique will provide greater coupling is strictly determined in a one- cal energy, however, is not simple, it is insight into the mechanisms underlying to-one fashion has attracted a great unlikely that multiple power strokes, the actomyosin molecular motor. deal of attention 3. Recently, the data each of which would be produced by that support the 1 :many variable some conformational change in myosin R~.ferences coupling model has accumulated not and/or actin, correspond to several i Woledge,R. C.. Curtin, N. A. and Homsher,E. ~1985)EnergeticAspects of Muscle only for the actomyosin motor (see Ref. transitions between the intermediate Contraction, AcademicPress 25 for a review and Ref. 26) but also for states of the ATPase cycle. In general, 2 Huxley,A. F. (1957) Prog. Biophys. Biophys. the gradual use of the energy stored in microtubule-based motors27. If the free Chem. 7,225-318 3 Huxley,H. E. (1990) J. Biol. Chem. 265, energy of hydrolysed ATP is subdivided a single activated state under strong 8347-8350 into small fractions available for several thermal agitation would seem unlikely 4 Yanagida,T., Nakase,M.. Nishiyama.K. and power strokes, the energy for each because of the collisions of water Oosawa.F. (1984) Nature 307, 58-60 power stroke will be several times molecules and vibrations of the atoms 5 Kron, S. J. and Spudich,J. A. (1986) Proc. Nat/ Acad. Sci. USA83, 6272-6276 smaller than that of a single ATP in the protein. This is one reason why

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TIBS 18 - SEPTEMBER1993 6 Toyoshima, ¥. Y. et aL (1987) Nature 328, 536-539 7 Kishino, A. and Yanagida, T. (1988) Nature 334, 74-76 8 Ishijima, A., Doi, T., Sakurada, K. and Yanagida, T. (1991) Nature 352, 301-306 9 Hotani, H. (1976) J. MoL BioL 106, 151-166 10 Inoue, S. (1981) J. Cell Biol. 89, 346-356 11 Morikawa, K. and Yanagida, M. (1981) J. Biochem. 89, 693-696 12 Harada, Y. and Yanagida, T. (1988) Cell Mot#. Cytoskel. 10, 71-76 13 Wieland, T. and Faulstich, H. (1978) CRC Crit. Biochem. 5, 185-260 14 Borejdo, J. and Morales, M. F. (1977) Biophys.

J. 20, 315-334 15 Iwazurni, T. (1987) Am. J. Physiol. 252, 253-262 16 Crawford, A. C. and Fettiplace, R. (1985) J. PhysioL 364, 359-379 17 Kamimura, S. and Kamiya, R. (1989) Nature 340, 476-478 18 Denk, W., Webb, W. W. and Hudspeth, A. J. (1989) Prec. Natl Acad. Sci. USA 86, 5371-5375 19 Sheetz, M. P., Turney, S., Qian, H. and Elson, E. L. (1989) Nature 340, 284-288 20 Kittel, C. (1964) Elementary Statistical Physics, Chap. 30, John Wiley & Sons 21 Huxley, H. E. (1969) Science 164, 1356-1366 22 Huxley, A. F. and Simmons, R. M. (1971) Nature 233, 533-538

23 Ebashi, S. (1991) Annu, Rev. Physiol. 53,1-16 24 Yanagida, T,, Arata, T. and Oosawa, F. (1985) Nature 316, 366-369 25 Burton, K. (1992) J. Muscle Res. Cell Mot#. 13,

590-607 26 Yanagida, T. (1993) Abs. 32nd Int. Physiol. Congress 7.4/0, 10 27 Taylor, E. W. (1993) Nature 361, 115-116. 28 Askin, A. and Dziedzic, J. M. (1987) Science

235, 1517-1520 29 Block, S. M. (1990) in Noninvasive Techniques in Cell Biology(Fosbett and Grinstein, eds), pp. 375-402, Wiley-Liss 30 Kuo, S. C. and Sheetz, M. P. (1992) Trends Cell 8ioL 2, 116-118

TALKINGPOINT TETANUS AND BOTULINUMneurotoxins are the most potent toxins known (mouse lethal dose <0.I ng kg-X). They are released by bacteria of the genus Clostridium as a single polypeptide chain of 150 kDa, later cleaved to generate two disulfide-linked fragments. The heavy chain (H, I00 kDa) is involved in cell binding and penetration, while the light chain (L, 50 kDa) is responsible for the intracellular activity~,~. The tremendous potency of these toxins is primarily due to their absolute neurospeclficity. They bind to the neuromuscular junction and are internalized inside vesicles3. Botullnum neurotoxln (BoNT, seven different serotypes: A-G) penetrates into the cytosol and blocks the release of acetylcholine, thus causing a flaccid paralysis. By contrast, tetanus neurotoxin (TeTx) migrates retroaxonally (in the reverse direction to nerve impu]ses) and, by transcytosis, reaches the spinal inhibitory interneurons, where it blocks neurotransmitter release with a consequent spastic paralysis R. Despite the opposing clinical symptoms of botulism and tetanus, these toxins act in a similar way at the cellular level. Since TeTx and BoNT are entirely responsible for the diseases tetanus and botulism, respectively, understanding their intracellular activity would also lead to the description of the molecular pathogenesis of these diseases. At the same time, we would expect to understand better the process of neuroexocytosis.

c. Monteeu©©oand G. S©hla¥oare at the Departmentof BiomedicalSciencesof the Universityof Padova,ViaTrieste75 1-35121, Padova,Italy. 324

• Tetanus and botulism neurotoxins: a new group of zinc proteases

The active forms of tetanus :,nd botulinum neurotoxins, released from the precursor molecule by specific proteolysis and reduction, block the release of neurotransmitters via a Zn2*-dependent protease activity. VAMP/synaptobrevin, an integral membrane protein of the synaptic vesicles, is cleaved at a single site by tetanus and botuliaum B, D and F neurotoxins. The unique sequence, mechanism of activation and site of activity of clostridial neurotoxins mark them out as an independent group of Zn2*-endopeptidases. Tetanus and botulinumneurotoxinsare Zna÷proteins The enormous potency of these neurotoxins, and analogy with other bacterial toxins with intracellular activity, suggests that TeTx and BoNT possess or induce a catalytic activity inside cells. Their amino acid sequences show a limited degree of similarity, concentrated mainly in the L chain. The segment with the closest homology is located in the central part and contains the Zn2*-binding motif, HEXXH,that is a characteristic feature of Zn2~endopeptidases 4"~2, Atomic absorption measurements showed that indeed TeTx and BoNT/A, B, C, E and F contain approximately one Zn2~ ion bound to the L chainS-7. As with other Zn~ proteases, the Zn2÷ion can be removed with EDTA or ortho-phenanthroline, thus forming

an apotoxin, and it can be replaced by incubation in ZnZ'-containing bufferss'7. The three-dimensional structures of four Zn2÷ endopeptidases have been solved by X-ray diffraction. In thermolysin 13, neutral protease t4 and Pseudomonas aeruginosa elastase L~, the Zn2~ ion is at the center of a tetrahedron, coordinated by the two histidine residues of the motif, by one water molecule that is bound to the glutamate residue of the motif, and by the carboxylate group of another glutamic acid residue. By contrast, the recently reported structure of astacin ~6,a Zn2~protease of the crayfish digestive tract, shows the Znz~ ion penta-coordinated by the two histidine residues, the water molecule that is bound to the glutamate residue of the motif, an additional histidine residue, and a tyrosine residue. On the basis of these findings, Zn2÷ endopeptidases

© 1993, ElsevierScience Publishers, (UK) 0968-0004/93/$06.00