NOTE: This page is ~1 year out of date; I hope to remedy that 'soon' (12-4-98) The vanadate ion In this crystal structure, ADP and VO4 are substituted for ATP. ATP would be hydrolyzed by myosin, and is therefore not sufficiently stable for the slow process of crystallization. ADP+VO4 mimics the structure of an ATP molecule undergoing hydrolysis. Note the closeness to the Gateway! In the BeF structure, a similar strategy was employed, except substituting BeF for VO4. Together, these bear a close structural resemblance to ATP; thus this state "precedes" the vanadate state in the stroking cycle. ADP Somewhat deceptive in these structures, the ADP is actually "in cahoots" with the vanadate, beryllium (Be) or other ions; the groups combine to simulate ATP (as in ADP.BeF structure), or ATP undergoing hydrolysis (as in ADP.vanadate or ADP.AlF structures) N29 Suppresses N483S Y34 Suppresses N483S N38 Suppresses L453F F85 F85S Suppresses N483S P128 P128Q Suppresses G680V E150 E150K is the 'supersuppressor': isolated multiple times as a suppressor of Y494K, W501L, L453F and N483S S165 S165L suppresses G691C G182: G182C is a suppressor of G680V. It is the third glycine residue in the famous P-loop sequence (important in contacting the phosphates of ATP) which spells GESGAGKT in myosins. G680A Suppressors While G680V was discovered in the original cs mutant hunt, we made G680A "by hand". The alanine side chain is a single methyl group (CH3), significantly less bulky than the side chain of valine (CH2(CH3)2). Indeed, the G680A mutant is less impaired than the G680V mutant. Similarly, we would predict that mutations that restore function to G680A would need to be less drastic than those required to revive G680V; this would appear to be the case: suppressors of G680A are more common and often cannot repair G680V. They nonetheless show some structural features similar to G680V suppressors. the Cluster: L175, L176, I177, (G182), T189, V192, I193 These positions represent a series of "solutions" to the defects introduced to the motor by the G680V mutation. As you can see in the figure, residues 175-177, 189, 192 and 453 (all shown as stick representations in green) form a tight grouping. The G182 position is parenthetical because despite being central in the primary sequence, it is not close to other listed members of the Cluster. Besides sharing a common location, Cluster members tend to have raised Basal ATPase and show poor activation of the ATPase by actin. On the other hand, L176I suppresses G691C L216 L216F was isolated as a common (6 instances total) suppressor of G680A or G680V. Characteristics very similar to members of the Cluster despite its location near the surface of the molecule. L222 L222F suppresses E531Q, and P536R, but apparently does NOT suppress R562H N235 N235D is a suppressor of G680V. Nearest noteworthy neighbor is R238, a member of the Gateway. G240 This position has turned up several times in our genetic screens. G240C and G240V were isolated as cs mutants in the original screen. G240N was isolated independently as a suppressor of G680V. G240C and G240V can also suppress G680V. It is conspicuously close in the sequence to R238, the positively charged member of the Gateway. G240V behaves similarly to many Cluster members biochemically. V334 V334F suppresses E531Q, P536R, and R562H. E360 E360Q was isolated as a suppressor of R562H Q407 Q407L Suppresses E531Q, P536R and R562H K413 K413R Suppresses E531Q and P536R, but not R562H D419 D419G suppresses E531Q and P536R G427 G427C suppresses E531Q, P536R and R562H L433 L433S suppresses E531Q, P536R and R562H V434 V434F Suppresses E531Q, P536R and R562H I455 I455T suppresses P536R The Gateway! R238, E459 These two residues potentially represent a critical part of the machinery regulating the release of Pi. Note how they form a tight association in the vanadate structure (which mimics a part of the cycle during which the phosphate is chemically separated from the rest of the ATP molecule), but are not as closely juxtaposed in the BeF structure. Yount et al. have conjectured that these residues mark one end of a tunnel through which Pi escapes during the myosin cycle. Since this path differs from the one used to enter the myosin molecule (the cave currently occupied by ADP), this would make myosin a "backdoor" enzyme. I point out that E459 interacts intimately with "the Camshaft", the helix running from 466-496, and that rotation of this helix accompanies the separation of the two members of the gateway. While neither of these residues has turned up in our mutant screens, we have isolated mutants at position 235 and 240, as well as many hits in the Camshaft itself. E467 E467Q was isolated as a cs mutant in the original screen; it lies close to the N-terminus of the camshaft. E476 This position was first studied as E476Q by Ruppel and Spudich, and turned up independently in our screen for suppressors of the G680V mutation (again as E476Q). Its most notable defects are the salt-sensitivity of its 'rigor' (no nucleotide) and +ADP actin binding states. L478 Note: this mutant originally appeared to be a suppressor of mutants in the Cluster, but these results have NOT BEEN REPRODUCIBLE. So for now, L478 is simply the point where the Cluster and the Camshaft come closest to contacting each other. Q480 Midway through the Camshaft, the Q480H change can suppress the W501L mutation. N483 Another G680V suppressor, this residue is 7 amino acids away from position 476, which puts them on the same "face" or side of the helix (Camshaft) in which they both lie. Like the E476Q alteration, the N483S change causes enhanced release of actin in the presence of salt and ADP. Unlike the E476Q change, N483S drastically impacts the biological performance of myosin. H485 H485R is a suppressor of Y494K M486 Another helix-dweller, the M486I change can restore function to the L453F change (a cluster member and suppressor of G680V). It can also suppress E531Q, P536R, R562H and DWrinkle mutants. It lies next to the 'bending point' in the CamShaft (466-496). K488 K488E suppresses G691C. L489 The amino acid at position 489 correlates with the one found at position 535, according to my viewing of myosin sequence alignments (see Phylogenetics for an explanation). E490 E490K suppresses both Y494K and G691C. Y494 One of the original cold-sensitive myosin mutations is Y494K, occurs near the endpoint of the CamShaft. This residue joins closely with 2 others from the cs screen: W501 and G691; not far away is yet another, G740. Clearly something about the structure of this region is critical, and its derangement drastically effects the functioning of the motor. Genetically, Y494K, N483S and W501L behave very similarly to one another, in that several other mutations will suppress each of the three (E150K, P650T, I656F, Y118F). L495 Next door neighbor to and suppressor of Y494K is L495P. The introduction of a proline (P) is noteworthy: proline is an oddly constructed amino acid that forces a specific conformation on the protein where it occurs. Somehow, such an alteration "reverses" the damage introduced by the Y494K change. K496 Another suppressor of Y494K is K496E. This change is one of the "bricks in the wall" in the case I am making for a direct, charge-based interaction between the region around amino acid 150 and amino acid 494. See the Sea of Hypotheses for more. W501 Another of the original cs mutants, and a member of the cs triplet cluster Y494K, W501L and G691C. These residues mark the end of the CamShaft. W501 is thought to be the tryptophan responsible for the change in fluorescence properties that myosin undergoes during its cycle. Fluorescence in response to certain wavelengths of light is a property of tryptophans in general, but the specific wavelength emitted by a tryptophan is dependent upon its local environment. Thus, during the myosin cycle the environment of some tryptophan (presumably this one) undergoes a significant alteration. D505 D505E suppresses G691C. G507 Yet another suppressor of Y494K is G507C. This is the closest we have yet come to genetically "marking" the ActBind helix (see below; the helix that follows the CamShaft terminates in a region of myosin that binds actin). One interpretation of this change is that the spatial relationship of the CamShaft to the ActBind helix is an important part of the workings of the motor. T513 The corresponding residue in rabbit myosin (C522), can be crosslinked to rabbit C707 (corresponding to Dicty T688) as reported by Ue. The ActBind Helix Amino acids 511-535 form a kinked a-helix that has its start after the turn that emerges from the Camshaft. This helix begins closely apposed to the Camshaft, but diverges as it progresses, and it terminates with residues thought to interact directly with actin. Several of the actin-interacting residues were mutated in the original cold-sensitive screen (E531Q, P536R, and nearby R562H, R562L and E586K). Despite strong predictions by myself that E476Q and N483S defects could be remedied by changes in this helix, we have been unable to gather any evidence for such an interaction. The "loopout" that drops down in the picture is intriguing, and sequence alignments suggest that this is a conserved feature amongst most or all myosins. We recently replaced the loopout with a single alanine, and have confirmed this change indeed blocks myosin function. Further experiments are underway The Wrinkle This apparently conserved 'flaw' in the ActBind (511-535) helix is delimited by glycine residues (G519 and G524) and contains a consecutive pair of prolines (P522 and P523). You might ask yourself: what is its purpose? And you might say to yourself: This is not my beautiful helix! We're currently fiddling with this region of myosin genetically; to date we know that replacement of the sequence G519-G524 with a single alanine gives a mutant phenotype; suppressors have been isolated and sequencing will follow shortly! L526 L526F suppresses G691C. E531 E531Q was isolated as a cold sensitive mutant S533 The corresponding residue in rabbit myosin is a cysteine (C540), which can be crosslinked to rabbit C697 (corresponding to Dicty C678) as reported by Chaussepied, Morales and Kassab. F535 The amino acid at position 535 correlates with the one found at position 489, according to my viewing of myosin sequence alignments (see Phylogenetics for an explanation). P536 P536R was isolated as a cold sensitive mutant E560 E560K suppresses N483S while E560R suppresses R562H R562 R562H and R562L were both isolated as cold sensitive mutants; their biological and biochemical defects do not appear to be very similar, but many mutations can suppress both R562H and R562L. A574 A574K Suppresses P536R D583 D583Y Suppresses of E531Q and R562H; D583N suppresses E531Q E586 E586K is one of the original cold-sensitive mutations; it also suppresses E531Q, P536R, R562H and DWrinkle mutations. QQ593KH The double mutant Q593K/Q594H suppresses delWrinkle L596 L596S suppresses P536R and R562H. L638 A "next door neighbor" of the Camshaft. Despite being more than 150 amino acids away in the primary sequence, this residue is nestled right up against the camshaft. The L638F change suppresses the G680V mutant. L645 L645I suppresses G691C T647 T647M is a suppressor of N483S. P650 Another "next door neighbor" of the Camshaft; changing this residue from Proline lifts the strong structural constraint introduced by the fact that the proline sidechain links back with the backbone portion of the amino acid, restricting the motion of the one relative to the other. The P650T mutation genetically counteracts mutations at positions 494, 501, and 483. I656 I656F suppresses N483S, Y494K and W501L. C678 The corresponding residue in rabbit myosin (C697), can be crosslinked to rabbitC540 (corresponding to Dicty S533) as reported by Chaussepied, Morales and Kassab. G680V: The Problem: This is the mutant that "started it all" in terms of our search for suppressors. We chose to focus on this member of our original collection of cold-sensitive mutants because of its notable location: two cysteine residues near position 680 are known to under significant conformational change during the motor's working cycle. Our characterization of the G680V mutant have led us to conjecture that the mutant motor "sticks" or pauses just after initial fruitful interaction with actin and at the point of phosphate release. This is a tremendously exciting and mysterious point in the cycle, where the motor is prepared to engage in its actual work--we think! T688 The corresponding residue in rabbit myosin (C707), can be crosslinked to rabbit C522 (corresponding to Dicty T513) as reported by Ue. G691 The G691C mutant was isolated as a cold-sensitive mutant. It's primary defect appears to be high basal ATPase, which can be attributed to promiscuous/premature release of phosphate. It's biological phenotype is proving difficult to reproduce; look at suppressors of this mutant with a somewhat jaundiced eye. G740 The G740D mutant is one of the original cs mutants.