Key Concepts. Learning Objectives

Transcription

1 Lectures 8 and 9: Protein Function, Ligand Binding -- Oxygen Binding and Allosteric Regulation in Hemoglobin [PDF] Reading: Berg, Tymoczko & Stryer, Chapter 7, pp problems in textbook: chapter 7, pp , #3,4,5,6,8 Updated on: 1/28/07 at 1:30 pm abbreviations used in this set of notes: Hb = hemoglobin, Mb = myoglobin Key Concepts Ligand binding is of fundamental importance 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. 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 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 the 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 the fractional saturation of the protein with O 2 is 0.5. In what part of the cooperative binding curve (what part of the [ligand] concentration range) is the protein predominantly in the conformation with low ligand binding affinity, and in what part of the ligand concentration Page 1 of 14

2 range is the predominant form the high binding affinity conformation? 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) the effect of the quaternary structural change on the 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? 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? 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 interactions) What kinds of interactions give the most SPECIFICITY in binding? 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 of other binding sites (on other subunits) of the same protein (Hb) molecule MYOGLOBIN (Mb) AND HEMOGLOBIN (Hb) MYOGLOBIN (Jmol structure of oxymb) (review from tertiary structure of proteins) functions: binds O 2 in muscle cells for a) storage, and b) intracellular transport uses a heme group (black, with purple Fe 2+ in fig. below) to bind O 2 Heme: an example of a prosthetic group Prosthetic group: a metal ion or an organic or metalloorganic compound other than an amino acid that is tightly bound to a protein polypeptide mostly α-helical -- 8 α helices, designated as helices A-H from N-terminus toward C-terminus 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, Tymoczko & Stryer, 6th ed. Figs. 2.48: and 2.49: Tertiary Structure and Distribution of Amino Acids in Myoglobin (hydrophobic residues in yellow, charged residues in blue, others in white) left: ribbon diagram (heme prosthetic group black, with purple Fe 2+ ); right, space-filling: A, surface view; B. cross-section through interior (hydrophobic residues in yellow, charged residues in blue, others in white) Page 2 of 14

3 Myoglobin monomeric (single polypeptide chain) just 1 O 2 binding site per molecule no communication possible between different Mb O 2 binding sites -- they're on different molecules Mb's O 2 binding non-cooperative -- no communication between different binding sites because each site is on a different molecule 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 P L complex [PL] in this expression are the equilibrium concentrations.) K association = 1/K dissociation FRACTIONAL SATURATION Y: fraction of total binding sites on the protein ([P] total ) OCCUPIED by ligand (Some books use "θ" instead of "Y" to symbolize fractional saturation.) Y = [occupied binding sites] / [total binding sites] units of Y minimum and maximum values of Y Approach to site saturation is asymptotic. hyperbolic plot for fractional saturation Y vs. [O 2 ] Page 3 of 14

4 Y = fractional saturation of a protein with a ligand, ratio of [occupied binding sites] / [total binding sites] Y vs. [O 2 ] same as [occupied sites]/[total sites] vs. po 2 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. HEMOGLOBIN (Jmol structure of deoxyhb) 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. relationship of biochemical (allosteric) control, understood at level of molecular structure, to physiology of whole organism Hemoglobin, a heterotetramer (quaternary structure α 2 β 2 ). structure of the 2 identical α subunits (red & pink in Fig. 7.5, below) very similar to structure of the 2 identical β subunits (yellow in Fig. 7.5)) both structures also very similar to structure of myoglobin (both 1 o and 3 o structure) Origin of different globin genes: single ancestral gene, with gene duplication and subsequent divergent evolution of sequences overall tertiary "fold" (motif with 8 α helices called the "globin fold") conserved for individual polypeptide chains. Hemoglobin's chains spontaneously assemble into 4 o structure 4 o structure stabilized by noncovalent bonds (no disulfide bonds in Mb or Hb) Hb structure = "dimer" of 2 αβ protomers Each α has one β "partner" with which it is more closely associated. allosteric 4 o structural changes (conformational changes): 4 o structure does NOT dissociate, but "α 1 β 1 " protomer shifts relative to "α 2 β 2 " protomer Conformational changes affect Hb's affinity for O 2 (see below) Berg, Tymoczko & Stryer, 6th ed. Fig. 7.5: Hemoglobin A (adult hemoglobin) quaternary structure showing "α 1 β 1 " and "α 2 β 2 " protomers Page 4 of 14

5 Hemoglobin tetrameric 4 O 2 binding sites per molecule binds O 2 COOPERATIVELY (different O 2 sites on same molecule communicate with each other) sigmoid plot of Y vs. po 2 Physiological function: WHY would Hb "want" its O 2 binding affinity regulated (cooperative binding), which higher affinity (tighter binding) at higher O 2 concentrations lower affinity (weaker binding) at lower O 2 concentrations? Answer lies in Hb function: transporting O 2 from lungs to tissues Page 5 of 14

6 2 aspects of transport: a) BINDING O 2 in lungs b) RELEASING O 2 in rest of tissues Berg, Tymoczko & Stryer, 6th ed Fig. 7.8: Cooperativity enhances O 2 delivery by hemoglobin. Sigmoid curve: Hb is ~ 98% saturated with O 2 in the lungs (where po 2 = ~100 torrs) 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. "Payload" of O 2 released in the tissues with the sigmoid curve is much greater (about 66% of the carrying capacity of the Hb) than the payload that would be released with a hyperbolic curve (about 38% of the carrying capacity). Structural basis for cooperative O 2 binding in hemoglobin O 2 binds to heme prosthetic group 1 heme/subunit = 1 O 2 binding site/subunit maximum of 4 O 2 can bind to Hb tetramer. 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"). Berg, Tymoczko & Stryer, 6th ed, p. 184: Heme structure Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed. Page 6 of 14

7 (2001), Fig. 5-5c: Binding of O 2 to myoglobin, showing the coordination of one O to the Fe 2+ of the heme and the hydrogen bond of the other O to the distal His (His E7) Note 2 hydrophobic residues, a Val and a Phe, which help keep Fe 2+ from oxidizing to Fe 3+ when oxygen is released Oxidation of iron would release oxygen as superoxide ion, O 2, a reactive oxygen species danger of oxidative damage to other cellular components. Oxidation of iron to Fe 3+ form of myoglobin or hemoglobin (the "met" form) inactivates the protein -- metmb and methb cannot bind O 2 ; the iron must remain reduced (Fe 2+ ) to function in O 2 binding and transport. Berg, Tymoczko & Stryer, 6th ed. Fig. 7.2: O 2 binding changes position of iron ion 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 (R group of 8th AA residue in sequence of the α helix called the "F" helix). Page 7 of 14

8 Berg, Tymoczko & Stryer, 6th ed. Fig. 7.14: Movement of F helix when O 2 binds to Hb. Movement of the heme iron upon O 2 binding pulls whole F helix toward the heme Conformational shift causes structural changes in interface between the two αβ protomers (α 1 β 1 and α 2 β 2 ). (OxyHb=red; deoxyhb = gray) Berg, Tymoczko & Stryer, 6th ed. Fig. 7.10: Hb conformational transition from deoxy conformation (T state) to oxy conformation (R state) "α 1 β 1 " protomer shifts relative to "α 2 β 2 " protomer Protomers rotate relative to each other by 15. T state predominates when no O 2 is bound (structure of deoxyhemoglobin) R state predominates when O 2 is bound (oxyhemoglobin). Animation showing overall changes (oxy conformation -- red O 2 bound to blue iron): (oxy5) Tertiary change (in ONE subunit): Shift from deoxy to oxy conformation deoxy Hb: Fe lies out of plane of heme ring when O 2 binds, Fe moves into plane of heme ring. (oxy1) Proximal His (His F8) is bound to Fe, His imidazole moves too, so F helix moves. (oxy2) Movement of F helix alters tertiary structure of that individual subunit. (oxy3) Quaternary change: Tertiary structural change affects interactions between subunits at interfaces. Page 8 of 14

9 (oxy4) Changes in interactions at protomer interfaces --> shift of entire tetramer from deoxy (T state) to oxy (R state) conformation, a quaternary structural change. (oxy5) Berg, Tymoczko & Stryer, 6th ed. Fig. 7.12: T to R transition. Think of observed O 2 binding curve for Hb as a combination of the binding curves that would be observed if all molecules remained in T state (weak binding hyperbolic curve, high P 50 ) or all molecules were in R state (tight binding hyperbola, low P 50 ). 2 theoretical models to explain cooperative ligand binding: concerted model and the sequential model Concerted model (= "MWC" model) Each O 2 that binds increases the probability that whole tetramer will switch from T to R state. Whole protein structure can exist in either the T state or the R state, but no "hybrid" quaternary structures occur with some subunits in T state and some subunits in R state. actually just the extremes of the sequential model (see below), with no "hybrid states" Berg, Tymoczko & Stryer, 6th ed. Fig. 7.11: Concerted model for allosteric ligand binding Sequential model Binding of ligand to one subunit changes conformation of subunit to which it binds, which induces changes in neighboring subunits that increase their binding affinity for ligand. allows for "hybrid" quaternary structures, with some subunits in R state and some in T state. Berg, Tymoczko & Stryer, 6th ed. Fig. 7.13: Sequential model for allosteric ligand binding Page 9 of 14

10 Allosteric behavior of hemoglobin isn't entirely concerted -- there are elements of sequential model involved in its behavior. 4 o structure doesn't change from T to R until there are at least 2 O 2 molecules bound to the tetrameric Hb, but individual subunits undergo 3 o changes when they bind O 2. 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 has a much higher O 2 binding affinity than Hb in 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! main allosteric inhibitor of O 2 binding: 2,3-bisphosphoglycerate (2,3-BPG). 2,3-BPG = a metabolic "byproduct" produced by isomerization of glycolytic intermediate 1,3-BPG in red blood cells. highly anionic structure glyceric acid = propionic acid (a 3-C carboxylic acid) with alcohol groups (OH) on carbons 2 and 3 those two OH groups are both esterified to phosphoric acid in 2,3-BPG. 2,3-BPG thus has 5 negative charges on a small molecule: 1 carboxylate - and 2 phosphate 2- groups: 5 negative charges on a small molecule! Where in Hb structure does 2,3-BPG bind, and by what type of interactions, and how does it reduce the O 2 binding affinity of Hb? 2,3-BPG binds to β chain residues in central cavity of the hemoglobin 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. Central cavity of T state of Hb (the deoxy conformational state) big enough for 2,3-BPG to fit 3 + charged groups from each β chain help bind 2,3-BPG by ionic interactions. Quaternary structural change from T to R state shrinks central cavity -- not enough room for 2,3-BPG to bind in R state Berg, Tymoczko & Stryer Fig. 7.16: 2,3-BPG binding to central cavity of hemoglobin (T state) Page 10 of 14

11 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 ). Fetal hemoglobin pregnant woman: O 2 taken in by mother through her lungs is transported 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. 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 and 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? ANSWER: Losing a + charge from binding site on protein would make 2,3-BPG bind less tightly. Having a lower fraction of Hb molecules with 2,3-BPG bound means more of fetal Hb is in R state (more than maternal Hb), so Under physiological conditions (at concentration 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. Page 11 of 14

12 Berg, Tymoczko & Stryer, 6th ed. Fig. 7.17: 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. 2 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. 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? 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".) 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". Berg, Tymoczko & Stryer, 6th ed. Fig. 7.21: 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): Page 12 of 14

13 proton concentration important because some groups have different pk a values in R state vs. 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. CO 2 binds to α amino groups of T state. Mutant human hemoglobins Mutant hemoglobins provide unique opportunities to probe structure-function 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 human hemoglobins -- properties are altered in one of the following ways: 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 a 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. 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? 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? zieglerm@u.arizona.edu Department of Biochemistry & Molecular Biophysics The University of Arizona Copyright ( ) 2007 All rights reserved. Page 13 of 14