Lectures 8 and 9 Protein Function: Ligand Binding and Allosteric Regulation

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1 Lectures 8 and 9 Protein Function: Ligand Binding and Allosteric Regulation Oxygen Binding to Myoglobin and Hemoglobin Allosteric Regulation of Hemoglobin Function Reading: Berg, Tymoczko & Stryer, 6th ed., Chapter 7, pp problems in textbook: chapter 7, pp , #3,4,5,6,8 abbreviations used in this set of notes: Hb = hemoglobin, Mb = myoglobin Jmol structure of myoglobin: Jmol structure of hemoglobin Jmol structure of hemoglobin with 2,3-bisphosphoglycerate (2,3-BPG) bound Key Concepts Ligand binding fundamentally important in biochemical phenomena. Heme (Fe protoporphyrin IX) in myoglobin and hemoglobin binds O 2 reversibly, without oxidation of the heme Fe +2 which is required for O 2 binding. Myoglobin and hemoglobin's structures and ligand binding properties have evolved differently for the different functions of the two proteins, and the structure-function relationships are very well understood. Mb is monomeric, 1 O 2 binding site per molecule, hyperbolic binding curve (no cooperativity). Hb is tetrameric, 4 O 2 binding sites per molecule, sigmoid binding curve indicative of cooperative ligand binding (structural communication between different binding sites by conformational changes). Hb is thus an allosteric protein. hemoglobin 1

2 Key Concepts, continued Hb is an allosteric protein. R state ("oxy" conformation, high O 2 binding affinity) stabilized by O 2 binding (O 2 is a homotropic effector) T state ("deoxy" conformation, low O 2 binding affinity) stabilized by binding of protons (H + ), CO 2, and/or 2,3-bisphosphoglycerate (2,3- BPG) (all heterotropic effectors, allosteric inhibitors) Allosteric regulation of O 2 binding to Hb is important to enhance the ability of Hb to RELEASE O 2 in the tissues. 2,3-BPG is needed in human erythrocytes (red blood cells) to reduce O 2 binding affinity enough to get effective release of O 2 in tissues. 2,3-BPG binds in central cavity of Hb (stoichiometry 1 BPG/Hb tetramer). Fetal Hb (HbF) has different quaternary structure from adult HbA (α 2 γ 2 vs. (α 2 β 2 ) Sequence difference between γ and β reduces HbF's affinity for 2,3-BPG, thus increasing its affinity for O 2 under physiological conditions. Learning Objectives Terminology: ligand, fractional saturation, prosthetic group, cooperativity, protomer, binding site, allosteric (allosteric site, allosteric effector, allosteric regulation) Briefly describe the tertiary structure of myoglobin and the hemoglobin subunits (the "globin fold"), explain how the helices are designated, and the roles of the proximal and distal His residues in heme and oxygen binding. Write a general protein-ligand binding/dissociation reaction in both the association and dissociation directions. What is the mathematical relationship between the association and dissociation equilibrium constants? Describe how and where in the structure of myoglobin and hemoglobin O 2 binds, including roles of protein functional groups and heme, and the oxidation state of the heme Fe required for O 2 binding. Sketch the O 2 binding curve [Y (fractional saturation) vs. po 2 ] for NONcooperative ligand binding to a protein, such as that for O 2 binding to myoglobin. On same plot, sketch a binding curve that shows cooperativity (cooperative ligand binding), such as that for O 2 binding to hemoglobin, and explain (again) what is meant by cooperativity. On both curves, indicate the value of P 50, the po 2 at which fractional saturation of protein with O 2 is 0.5. In what part of the cooperative binding curve (what part of the [ligand] concentration range) is protein predominantly in conformation with low ligand binding affinity, and in what part of the ligand concentration range is the predominant form the high binding affinity conformation? hemoglobin 2

3 Learning Objectives, continued Explain how hemoglobin works physiologically (in vivo), i.e., how cooperativity in O 2 binding to hemoglobin facilitates loading of O 2 in the lungs and unloading of O 2 in the tissues. Include the role of the R state (oxy conformation) and the T state (deoxy conformation) of hemoglobin. Briefly describe the structural change that occurs when O 2 binds to the heme of a subunit of hemoglobin, including a) what in the heme structure triggers the protein structural change when O 2 binds, b) how that first protein structural change is communicated to other subunits to change the quaternary structure and the O 2 binding affinity of the other subunits, and c) effect of the quaternary structural change on size of the central cavity. Explain the effect of 2,3-bisphosphoglycerate on the affinity of mammalian hemoglobin for oxygen, and describe where on the hemoglobin molecule 2,3-BPG binds, how many molecules of 2,3-BPG bind to one hemoglobin tetramer, and predominantly by what type of noncovalent interactions the 2,3-BPG is bound. Does 2,3-BPG bind to the R state or the T state of hemoglobin? Learning Objectives, continued Explain why maternal red blood cells release O 2 and fetal red blood cells bind O 2 in the placenta in terms of a) the difference in protein primary structure and quaternary structure (subunit composition) between HbA (adult, maternal) and HbF (fetal hemoglobin), b) the effect of the structure of HbF on its 2,3-BPG binding affinity compared to HbA,and c) the resultant difference in O 2 binding properties of the 2 hemoglobins and its physiological significance. Discuss the Bohr effect (H + binding and CO 2 binding), in terms of a) the effect of increasing concentrations of either of these ligands on the O 2 binding curve for hemoglobin, b) the physiological significance of this phenomenon. For a mutant in which the T-R equilibrium is shifted toward the R state, what type of change would you expect in P 50? Does that mean O 2 affinity of mutant is higher or lower than normal? For a mutant in which the T-R equilibrium is shifted toward the T state, what type of change would you expect in P 50? Does that mean O 2 affinity of mutant is higher or lower than normal? hemoglobin 3

4 Ligand Binding The essence of protein function/action is BINDING (recognition of and interaction with other molecules). BINDING: result of specific, usually NONCOVALENT interactions between molecular surfaces SHAPE complementarity (lots of van der Waals interactions) CHEMICAL complementarity (hydrogen bonds, salt linkages) HYDROPHOBIC EFFECT (hydrophobic ligand minimizes exposure to water by binding in hydrophobic site in protein) What kinds of interactions give the most SPECIFICITY in binding? Terminology: LIGAND: a molecule or ion (usually small) that's bound by another molecule (usually large, e.g., a protein) COOPERATIVE ligand binding ("cooperativity"): binding of a ligand (O 2 ) to 1 binding site affects the properties (binding affinities) of other binding sites (on other subunits) of the same protein molecule. Example: O 2 binding to Hb General case: ligand binding to a protein chemical equation for dissociation of ligand (L) from protein (P): equilibrium dissociation constant K d for reaction: Concentrations of free protein (empty binding sites) = [P], free ligand [L], and PL complex [PL] in this expression are the equilibrium concentrations. K association = 1/K dissociation hemoglobin 4

5 FRACTIONAL SATURATION Fractional saturation = Y = fraction of total binding sites on protein ([P] total ) occupied by ligand Y = [occupied binding sites] / [total binding sites] = Plot of fractional saturation Y vs. [ligand] is hyperbolic. Approach to site saturation is asymptotic. Fractional Saturation: equation for a rectangular hyperbola units of Y? minimum and maximum values of Y? Plot of Y vs. [ligand]: K d = concentration of ligand needed to HALF-SATURATE the binding sites When Y = 0.5, [L] = K d Suppose you have 2 proteins that both bind the same ligand, one with K d = 10 5 M and the other with K d = 10 7 M. Which one has the higher binding affinity for the ligand? hemoglobin 5

6 Myoglobin (Mb) (REVIEW from tertiary structure) Jmol structure of Mb: binds O 2 in muscle cells for storage and for intracellular transport, using a heme group mostly (70%) α-helical; rest mostly turns & loops (at surface) first high-resolution crystal structure of a protein ever determined very compact structure (almost no empty space inside) very water-soluble 5 Pro residues, 4 in turns 8 α helices, designated by letters A - H, from N to C terminus Helices amphipathic (surface sides hydrophilic R groups, buried sides more hydrophobic R groups) Myoglobin structure Berg et al., Fig. 2.48B; heme black with purple Fe 2+ Nelson & Cox, Lehninger Principles of Biochemistry, Fig (heme in red; blue residues: Leu, Ile, Val, Phe) hemoglobin 6

7 Myoglobin structure, continued Distribution of Amino Acids in Mb structure (hydrophobic residues in yellow, charged residues in blue, others in white) A. surface view ; B. cross-sectional view showing interior of protein NOTE: many charged residues on surface, none in interior many hydrophobic residues in interior, but also a few on surface The only polar residues inside are 2 His residues involved in binding the heme and O 2. Berg et al., Fig O 2 binds to heme prosthetic group of Mb (and Hb) Heme: iron protoporphyrin IX, with bound Fe 2+ 1 heme per Mb = 1 O 2 binding site per Mb molecule 1 heme per Hb subunit, so maximum of 4 O 2 can bind to Hb tetramer. 6 coordination positions of heme Berg et al., p.184 Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 5-1 hemoglobin 7

8 Heme Fe 2+ coordination 4 positions to 4 N atoms in heme (all close to being in same plane) 5th coord. bond to N in a His residue in the protein (His F8, the "proximal His"). 6th coordination position is to O 2 (when O 2 binds) O 2 binds between Fe 2+ and another His in protein (HisE7, the "distal His"). 2 hydrophobic residues, Val and Phe, help keep Fe 2+ from becoming oxidized to Fe 3+. Binding of O 2 to myoglobin, showing coordination of one O to the Fe 2+ of the heme and hydrogen bond of the other O to distal His (His E7) Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 5-5c distal His, part of E helix (hydrogen bond to distal O atom) proximal His, part of F helix (attached to heme Fe 2+, so if Fe 2+ moves, F helix moves) O 2 binding by Myoglobin Myoglobin monomeric (single polypeptide chain) just 1 O 2 binding site per molecule Mb's O 2 binding non-cooperative -- no communication is possible between different binding sites because each site is on a different molecule. Plot of Y vs. po 2 is hyperbolic. P 50 is formally equivalent to K d, the ligand conc. (po 2 ) when Y = 0.5. hemoglobin 8

9 Plot of fractional saturation (Y) vs. po 2 for Mb po 2 (O 2 concentration) in pressure units (torr) 1 torr = 1 mm Hg at 0 C and standard gravity, i.e. sea level. P 50 = ligand (O 2 ) conc. in pressure units when Y = 0.5 (50% saturation) Mb function (O 2 storage and transport within cells, like a little molecular "bucket brigade") requires no regulation. O 2 binding by myoglobin (Berg et al. Fig. 7-6) ( = P 50 ) Suppose a mutant myoglobin has a P 50 of 5 torr. Is its O 2 binding tighter or weaker than that of normal Mb (shown on graph)? Hemoglobin, a heterotetramer (α 2 β 2 ) Jmol structure O 2 transport protein very well-understood example of allosteric regulation, important concept in regulation of activity of many enzymes as well O 2 binding to hemoglobin is cooperative. Biochemical (allosteric) control, understood at level of molecular structure, related to physiology of whole organism 2 identical α subunits (red) structurally similar to 2 identical β subunits (yellow) α and β also very similar to structure of myoglobin (both primary and tertiary structure) gene duplication of single ancestral gene and subsequent divergent evolution of sequences --> different globin genes tertiary "fold" ( globin fold ) conserved through evolution Berg et al., Fig hemoglobin 9

10 Tetrameric Quaternary Structure of Hb: α 2 β 2 = (αβ) 2 Hemoglobin's chains spontaneously assemble into quaternary structure. Quaternary structure stabilized by noncovalent bonds (no disulfide bonds in Mb or Hb) Hb structure = a "dimer" of 2 αβ protomers: (αβ) 2 α 1 β 1 + α 2 β 2 Each α has one β "partner" with which it is more closely associated. Conformational changes in tetramer affect Hb's affinity for O 2. Berg et al. Fig. 7-5 Binding of O 2 to Hb is sigmoid, not hyperbolic. Sigmoid binding curve (Y vs. [ligand]) Indicates that there are multiple interacting binding sites for ligand on each protein molecule -- the 4 different sites communicate with each other (cooperative binding, allosteric effects). Mb has 1 O 2 binding site per molecule, so no opportunity for interaction (no communication between binding sites) -- hyperbolic binding curve, noncooperative binding. Berg et al. Fig. 7-7 hemoglobin 10

11 Oxygen binding -- Hemoglobin Allosteric protein (Greek, allos = other ; stereos = shape ) (def.) Binding of ligand to one site on (multisubunit) protein affects the binding properties of another site on same protein molecule. Hb: 4 O 2 sites per Hb tetramer sigmoid binding curve, diagnostic of cooperative binding ( cooperativity ) communication between different ligand binding sites on same multimeric protein molecule Communication occurs via structural (conformational) changes. 2 interconvertible conformational states of Hb, T state and R state The sigmoid O 2 binding curve of Hb -- a composite Low affinity curve at low O 2 conc. (T state predominating) High affinity curve at high O 2 conc. (R state predominating) T state (low O 2 affinity) <==> R state (high O 2 affinity) In absence of O 2, equilibrium lies far toward T state (weak O 2 binding). In presence of O 2, equilibrium is shifted toward R state (tight binding). O 2 binding is a molecular switch that induces a change in O 2 binding affinity over the whole population of Hb molecules in solution. Berg et al. Fig hemoglobin 11

12 Physiological reason for regulation of O 2 binding of Hb Why would Hb want to regulate the tightness of its O 2 binding, such that because of a conformational change, the protein binds O 2 with higher affinity (tighter binding) at higher O 2 concentrations lower affinity (weaker binding) at lower O 2 concentrations. Explanation lies in O 2 transport function of Hb. 2 aspects of transport: a) binding O 2 in lungs b) releasing O 2 in rest of tissues Cooperativity enhances O 2 delivery/release by Hb. Cooperativity enhances O 2 delivery/release by Hb. Sigmoid curve: Hb is ~ 98% saturated with O 2 in the lungs (where po 2 = ~100 torrs) At high O 2 conc., both Mb and Hb are essentially saturated with O 2. Difference is clear at lower O 2 conc. in tissues, where Mb stays loaded, doesn t unload much O 2. With a sigmoid curve Hb can UNLOAD (release, dissociate) more of its carrying capacity of O 2 in the tissues (where po 2 = ~20 torr) than it could release with non-cooperative binding. Berg et al., Fig. 7-7 hemoglobin 12

13 Structural basis for cooperativity in Hb O 2 binding changes position of Fe 2+ in heme of Hb, initiating structural changes. In absence of bound O 2, heme iron lies slightly outside porphyrin plane, bound (coordinated) to an N of a His residue, the proximal His (His F8). When O 2 binds, Fe 2+ moves into plane of heme, pulling with it His F8 residue. Tertiary structural changes in T --> R O 2 binding pulls proximal His (F8) toward heme, moving F helix. Conformational change in one subunit causes structural changes in interface between α 1 β 1 and α 2 β 2 protomers, eventually triggering quaternary change in whole Hb tetramer. (OxyHb = red; deoxyhb = gray) Berg et al. Fig hemoglobin 13

14 Quaternary structural changes in T --> R α 1 β 1 protomer shifts relative to α 2 β 2 protomer and rotates ~15. Figure is looking down 2-fold symmetry axis, through central cavity (β subunits yellow). Note decrease in size of central cavity in R state compared to T. Berg et al. Fig Animations showing T --> R conformational changes as O 2 binds (R state has (red) O 2 bound.) Overall change (summary): Note change in size of central cavity! Fe 2+ moves into plane of heme when O 2 binds: Proximal His moves with iron, so F helix moves: Movement of F helix alters tertiary structure of that individual subunit: Tertiary changes in individual subunits cause structural changes in protomer interfaces (between α 2 and β 1 and between α 1 and β 2 ): When at least 1 O 2 has bound to each αβ protomer (so at least 1 subunit per protomer has changed tertiary conformation), whole quaternary structure shifts: hemoglobin 14

15 Any condition that shifts the R T equilibrium toward the R state increases the O 2 binding affinity. Any condition that shifts the R T equilibrium toward the T state decreases the O 2 binding affinity. 3 allosteric inhibitors of O 2 binding "tune" the O 2 affinity of hemoglobin 2,3-bisphosphoglycerate (2,3-BPG) protons (H + ) carbon dioxide (CO 2 ) Why are allosteric inhibitors of O 2 binding needed? Purified human Hb -- much higher O 2 binding affinity than Hb in erythrocytes (red blood cells). Without some negative allosteric regulator to reduce its affinity for O 2, human Hb wouldn't be able to unload much O 2 at all in the tissues. Hb would release only about 8% of its payload at 20 torr! 2,3-bisphosphoglycerate (2,3-BPG) main allosteric inhibitor of O 2 binding to human Hb 2,3-BPG = metabolic "byproduct" produced by isomerization of glycolytic intermediate 1,3-BPG in red blood cells highly anionic structure: glycerol: CH 2 OH CHOH CH 2 OH glyceric acid = propionic acid (a 3-C carboxylic acid) with OH groups on C2 and C3 C2 and C3 OH groups esterified to phosphates in 2,3-BPG Where in Hb structure does 2,3-BPG bind? By what type of interactions? How does it reduce the O 2 binding affinity of Hb? hemoglobin 15

16 2,3-BPG binds to β chain residues in central cavity of Hb tetramer. Jmol structure of BPG-Hb stoichimetry of 2,3-BPG binding = 1 BPG per Hb tetramer. 2,3-BPG does NOT bind where the O 2 binds. 3 + charged groups from each β chain in central cavity help bind 2,3- BPG by ionic interactions. Berg et al. Fig Central cavity of T state of Hb (the deoxy conformational state) big enough for 2,3-BPG to fit Quaternary structural change from T to R state shrinks central cavity -- not enough room for 2,3-BPG to bind in R state s central cavity 2,3-BPG binds only to T state, stabilizing T state, and shifting equilibrium toward T (weak O 2 binding form) and away from R, so whole sigmoid O 2 binding curve is shifted to higher O 2 concentrations (weaker O 2 binding, higher P 50 ). like Berg et al., Fig but in ribbon diagram (deoxyhb) (oxyhb) hemoglobin 16

17 Fetal Hemoglobin pregnant woman: O 2 taken in by mother through her lungs is transported by maternal adult Hb (α 2 β 2 ) to placenta for delivery to fetus. In placenta, maternal (adult) Hb must release O 2, and fetal Hb must bind O 2. For effective transfer, fetal Hb must be able to bind O 2 more tightly than maternal Hb. How does fetal Hb manage to bind O 2 tighter than maternal Hb? Different globin genes expressed at different times in embryonic development encoding different Hb subunits, with O 2 binding properties tailored to embryo's needs at that stage Last ~2/3 of fetal life: predominant form of Hb present is α 2 γ 2. γ chains are being made rather than β chains. β vs. γ -- similar AA sequences, but crucial differences: β chains have His 143, in 2,3-BPG binding site. γ chains have Ser 143, in 2,3-BPG binding site. What would be the effect of losing a + charged group from BPG binding site, and how would that affect O 2 binding affinity? Fetal Hemoglobin, continued A lower fraction of Hb molecules with 2,3-BPG bound means more of fetal Hb is in R state (more than maternal Hb). Thus, under physiological conditions (at conc. of 2,3-BPG found in erythrocytes) fetal Hb has a higher O 2 binding affinity than maternal Hb. Thus mother can "deliver" O 2 to fetus. O 2 affinity of fetal red blood cells Fetal Hb binds O 2 more tightly than maternal (adult) Hb because fetal Hb binds 2,3-BPG less tightly than adult Hb does. Berg et al. Fig hemoglobin 17

18 Other negative regulators of O 2 binding to Hb: H + and CO 2 Binding of protons and binding of CO 2 promote release of O 2 (weaker binding of O 2 ). Protons and CO 2 preferentially bind to the T state of Hb shift T R equilibrium toward T state reduce O 2 binding affinity of Hb. Effect of H + and CO 2 (to promote release of O 2 from Hb): the "Bohr effect" Why is it useful physiologically that protons and CO 2 bind more tightly to T state than to R state? Physiological role of H + and CO 2 as negative allosteric effectors of O 2 binding to hemoglobin Lungs: (Hb "wants" to bind O 2 tightly, to "load up".) ph is high" (ph ~7.4) ([H + ] is low) [CO 2 ] is low because it's being gotten rid of (exhaled) [O 2 ] is high. Ligand conc. conditions all favor R state. Result: O 2 binds tightly. (That's what you want, to BIND O 2, maximal "loading in the lungs.) Tissues: (Hb "wants" to dissociate its O 2, to UNLOAD) ph is low (ph ~7.2) ([H + ] is high) because catabolism (breakdown of nutrients) produces protons (acid, especially lactic acid in active muscle tissue) [CO 2 ] is high because CO 2 = end product of oxidation of C atoms in catabolism of nutrients. [O 2 ] is low. Ligand concentration conditions all favor T state. Result: O 2 binds weakly. (That's what you want, to DISSOCIATE O 2, maximal "unloading". hemoglobin 18

19 Effect of ph and of CO 2 on O 2 binding affinity of Hb (the Bohr effect). Chemical basis of Bohr effect (at least partially understood): CO 2 binds to α amino groups of T state. Proton concentration: some groups have different pk a values in R state from their pk a values in T state: higher pk a s in T state. Higher pk a in T state means that ionizable group binds protons more tightly in T state than in R state. In the T state, when those groups are protonated, they can form salt links with charged groups that can t form in R state. Those salt links stabilize T conformation. Berg et al., Fig Mutant human hemoglobins Mutant hemoglobins provide unique opportunities to probe structurefunction relations in a protein. There are nearly 500 known mutant hemoglobins and >95% represent single amino acid substitutions. About 5% of the population carries a variant hemoglobin. Some mutant hemoglobins cause serious illness. The structure of hemoglobin is so delicately balanced that small changes can render the mutant protein nonfunctional. 4 types of mutant hemoglobins Properties are altered in one of the following ways: 1. Mutation in heme binding pocket leads to loss of heme. 2. Mutation disrupts tertiary structure of a subunit. 3. Mutation stabilizes methemoglobin (Fe 3+ oxidation state of heme in Hb). 4. Mutation stabilizes the R state, or stabilizes the T state, compared to their stabilities in normal HbA. hemoglobin 19

20 4 types of mutant hemoglobins 1. Mutation in heme binding pocket leads to loss of heme. produces a nonfunctional protein (can't bind O 2 ) 2. Mutation disrupts tertiary structure of a subunit. produces protein with reduced stability or impaired function or both 3. Mutation stabilizes methemoglobin (Fe 3+ oxidation state of heme in Hb). In order for hemoglobin to reversibly transport O 2, iron must remain in ferrous (Fe 2+ ) state. Oxidizing iron to Fe 3+ produces methb, which does not transport O 2. Red blood cells contains enzymes that can re-reduce the iron in the occasional normal HbA molecule whose iron gets oxidized. Mutations that stabilize methb provide a negatively charged oxygen atom as a ligand for the iron, e.g., Glu instead of the normal distal His imidazole N. Negatively charged oxygen ligand stabilizes iron in the Fe 3+ state. Mutant hemoglobins, continued 4. Mutation stabilizes the R state, or stabilizes the T state, compared to their stabilities in normal HbA. Mutations at the subunit interfaces between the two αβ protomers often interfere with quaternary structure of hemoglobin. Such mutations can change the relative stabilities of hemoglobin's R and T states, shifting the equilibrium more toward R or more toward T state, thereby affecting O 2 affinity of mutant hemoglobin. Normal HbA has P 50 = 26 mm Hg (= 26 torr, or about 3.5 kpa). [P 50 is the po 2 at which the fractional saturation = 0.5, so half of Hb's O 2 binding sites are occupied.] For a mutant in which the T-R equilibrium is shifted toward the R state, what type of change would you expect in P 50? Does that mean O 2 affinity of mutant is higher or lower than normal? Would the P 50 for the mutant be higher or lower than normal? For a mutant in which the T-R equilibrium is shifted toward the T state, what type of change would you expect in P 50? Does that mean O 2 affinity of mutant is higher or lower than normal? Would the P 50 for the mutant be higher or lower than normal? hemoglobin 20