CHAPTER 24 – Evolution of Protein Molecules

The ability of fibronectin to bind cells can be accounted for by the tetrapeptide L-arginyl-glycyl-L-aspartyl-L-serine, a sequence which is part of the cell attachment domain of fibronectin and present in at least five other proteins. This tetrapeptide may constitute a cellular recognition determinant common to several proteins.

Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule

Lectins: Carbohydrate-Specific Proteins That Mediate Cellular Recognition.

Ca(2+) regulation of contraction in vertebrate striated muscle is exerted primarily through effects on the thin filament, which regulate strong cross-bridge binding to actin. Structural and biochemical studies suggest that the position of tropomyosin (Tm) and troponin (Tn) on the thin filament determines the interaction of myosin with the binding sites on actin. These binding sites can be characterized as blocked (unable to bind to cross bridges), closed (able to weakly bind cross bridges), or open (able to bind cross bridges so that they subsequently isomerize to become strongly bound and release ATP hydrolysis products). Flexibility of the Tm may allow variability in actin (A) affinity for myosin along the thin filament other than through a single 7 actin:1 tropomyosin:1 troponin (A(7)TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca(2+) binding sites on TnC, conformational changes resulting from Ca(2+) binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca(2+) binding to TnC enhances TnC-TnI interaction, weakens TnI attachment to its binding sites on 1-2 actins of the regulatory unit, increases Tm movement over the actin surface, and exposes myosin-binding sites on actin previously blocked by Tm. Adjacent Tm are coupled in their overlap regions where Tm movement is also controlled by interactions with TnT. TnT also interacts with TnC-TnI in a Ca(2+)-dependent manner. All these interactions may vary with the different protein isoforms. The movement of Tm over the actin surface increases the "open" probability of myosin binding sites on actins so that some are in the open configuration available for myosin binding and cross-bridge isomerization to strong binding, force-producing states. In skeletal muscle, strong binding of cycling cross bridges promotes additional Tm movement. This movement effectively stabilizes Tm in the open position and allows cooperative activation of additional actins in that and possibly neighboring A(7)TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca(2+) binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca(2+) regulates the strong binding of M.ADP.P(i) to actin, which precedes the production of force (and/or shortening) and release of hydrolysis products. 3) The initial rate of force development depends mostly on the extent of Ca(2+) activation of the thin filament and myosin kinetic properties but depends little on the initial force level. 4) A small number of strongly attached cross bridges within an A(7)TmTn regulatory unit can activate the actins in one unit and perhaps those in neighboring units. This results in additional myosin binding and isomerization to strongly bound states and force production. 5) The rates of the product release steps per se (as indicated by the unloaded shortening velocity) early in shortening are largely independent of the extent of thin filament activation ([Ca(2+)]) beyond a given baseline level. However, with a greater extent of shortening, the rates depend on the activation level. 6) The cooperativity between neighboring regulatory units contributes to the activation by strong cross bridges of steady-state force but does not affect the rate of force development. 7) Strongly attached, cycling cross bridges can delay relaxation in skeletal muscle in a cooperative manner. 8) Strongly attached and cycling cross bridges can enhance Ca(2+) binding to cardiac TnC, but influence skeletal TnC to a lesser extent. 9) Different Tn subunit isoforms can modulate the cross-bridge detachment rate as shown by studies with mutant regulatory proteins in myotubes and in in vitro motility assays. (ABSTRACT TRUNCATED)

https://www.physiology.org/doi/pdf/10.1152/physrev.2000.80.2.853

Regulation of Contraction in Striated Muscle

The x-ray crystal structure of a peptide corresponding to the leucine zipper of the yeast transcriptional activator GCN4 has been determined at 1.8 angstrom resolution. The peptide forms a parallel, two-stranded coiled coil of alpha helices packed as in the "knobs-into-holes" model proposed by Crick in 1953. Contacts between the helices include ion pairs and an extensive hydrophobic interface that contains a distinctive hydrogen bond. The conserved leucines, like the residues in the alternate hydrophobic repeat, make side-to-side interactions (as in a handshake) in every other layer of the dimer interface. The crystal structure of the GCN4 leucine zipper suggests a key role for the leucine repeat, but also shows how other features of the coiled coil contribute to dimer formation.

https://science.sciencemag.org/content/sci/254/5031/539.full.pdf

X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil.

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum.

Regulation of contraction in skeletal muscle occurs through calcium binding to the protein troponin C. The solution structures of the regulatory domain of apo and calcium-loaded troponin C have been determined by multinuclear, multidimensional nuclear magnetic resonance techniques. The structural transition in the regulatory domain of troponin C on calcium binding involves an opening of the structure through large changes in interhelical angles. This leads to the increased exposure of an extensive hydrophobic patch, an event that triggers skeletal muscle contraction.

Structures of the troponin C regulatory domains in the apo and calcium-saturated states.

Tropomyosin: Amino Acid Sequence and Coiled-Coil Structure

Structural interpretation of the two-site binding of troponin on the muscle thin filament

A tentative amino-acid sequence for the COOH-terminal half of rabbit skeletal tropomyosin is reported. These studies confirm our previous conclusions that this tropomyosin consists of several different but similar polypeptide chains. In the sequence, nonpolar residues occur in two series at intervals of seven residues. Amino-acid residues in series I are three residues on the NH2-terminal side of, and four residues on the COOH-terminal side of, residues in series II. The presence of occasional charged or ambivalent residues in the positions of series I or II does not lead to a disruption of this long-range pattern. The majority of residues located between the nonpolar residues are charged or polar amino acids. Two highly similar or identical α-helices with the reported sequence can be packed together in parallel in a coiled-coil structure. These may be in register or staggered by seven residues or some multiple of it. The observation that groups of small hydrophobic side chains appear to alternate with groups of bulky side chains suggests that a staggered arrangement of the two α-helices would maximize the regularity and hydrophobic interactions of the coiled-coil. Model building considerations show that this would occur with a stagger of 14 residues. Such an arrangement could account for the end-to-end aggregation of tropomyosin in solution, and in crystal and tactoid filaments. However, a structure in which the two polypeptides are in register cannot be ruled out.

https://www.pnas.org/content/pnas/69/12/3800.full.pdf

Lawrence B. Smillie

Papers

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum.

Structures of the troponin C regulatory domains in the apo and calcium-saturated states.

Tropomyosin: Amino Acid Sequence and Coiled-Coil Structure

Structural interpretation of the two-site binding of troponin on the muscle thin filament

Amino-acid sequence of rabbit skeletal tropomyosin and its coiled-coil structure.