Chapter 3: Binding and Molecular Recognition

The receptor protein can readily

distinguish even minor differences in
its target, such as the substitution of
an isoleucine for a leucine.
 Learning Goals

By the end of this chapter, you should be able to:

• Explain in qualitative terms how the interaction between a receptor and a ligand depends on the
concentration of the ligand.

• Describe the key principles for the binding of oxygen by myoglobin and hemoglobin.

• Discuss the concept of binding cooperativity and how it applies to hemoglobin, and how cooperativity
enhances the ability of hemoglobin to function as a highly effective oxygen carrier.

• Describe some key binding proteins in the immune system, including toll-like receptors, immunoglobulins,
and major histocompatibility complex proteins, and how binding reactions involving these proteins function in
the immune system.

• Calculate parameters of binding reactions using the concentrations of the reactants and the characteristics
such as the dissociation constant and kinetic rate constants
 Outline: Selective Power

3.1 Binding Is a Fundamental Process in Biochemistry

3.2 Myoglobin Binds and Stores Oxygen

3.3 Hemoglobin Is an Efficient Oxygen Carrier

3.4 The Immune System Depends on Key Binding Proteins

3.5 Quantitative Terms Can Describe Binding Propensity

 3.1 Binding Is a Fundamental Process in Biochemistry

Molecular recognition refers to the specific interaction between two or more

molecules, where one molecule (receptor) identifies and binds to a specific
target molecule (ligand). This process is driven by non-covalent interactions
such as hydrogen bonding, van der Waals forces, electrostatic interactions, and
hydrophobic effects.

Molecular recognition is fundamental in biological systems, playing a key role

in processes like enzyme-substrate binding, antigen-antibody interactions, and
DNA-protein recognition.
 Binding depends on the concentrations of the binding partners

A characterizing parameter is the concentration of L ( [L] ) at which half of the

receptor is bound to L and half is free. This is designated as L1/2.
The binding of testosterone to the estrogen receptor is very
weak with L1/2 > 1μM, more than 1000 times larger than that
for estradiol.
The protein has a narrow binding pocket lined with hydrophobic
residues such as leucine and phenylalanine that fits neatly around
the ligand but would likely sterically clash with thicker ligands such
as testosterone.

Estrogen receptor include

binding pockets,
complementary to the
shapes of specific ligands
Two Synthetic Ligands for Estrogen receptors

A factor of more than 20.

 Binding is a dynamic process

Dissociation and association are constantly occurring, even at

equilibrium where the concentrations of all species are constant.
The structure of myoglobin. Heme comprises 4 pyrrole
Heme functions rings that uses their nitrogen
as the oxygen-binding site. atoms to bind to Fe.

Oxygen storage protein

 Half saturation of human myoglobin occurs
when the partial pressure of oxygen is 2 torr.

cf, @sea level, 150 torr (mm Hg)

Structural Change upon binding of O2.
Sixth site

Ferrous (Fe2+) oxidation states

Oxygen binding changes the position of the Two resonance structures in the complex
heme iron ion. In deoxymyoglobin, the iron ion between iron and oxygen in myoglobin.
lies slightly outside the plane of the porphyrin and One resonance structure has Fe2+ bound to
moves into the plane of the porphyrin upon dioxygen, and the other has Fe3+ bound to
oxygen binding. superoxide ion.
Preventing the release of superoxide Superoxide is less likely to be
released due to two reasons.

1. Radical damage
2. Ferric state producing
malfunctioning metmyoglobin

hydrogen
bond
imidazole

cf, metmyoglobin reductase

Bound oxygen is stabilized through a hydrogen bond between the distal histidine
and one of the oxygen atoms. The protein component of myoglobin controls the
intrinsic reactivity of heme, making it more suitable for reversible oxygen binding.
 Carbon monoxide (CO) binding state

carbon monoxide poisoning

Binding studies indicate that deoxymyoglobin binds CO very tightly, with a P50
of approximately 0.02 torr, a hundredfold tighter than oxygen.
3.3 Hemoglobin Is an Efficient Oxygen Carrier
 Tetrameric Structure of Hemoglobin

4 peptides:
two of (αβ heterodimer)

Iron-iron distance
Ranging from 24 to 40Å

Max Perutz, horse heart 1A3N.pdb

• Hemoglobin a Chain vs. Myoglobin : 25% Identity

• Hemoglobin b Chain vs. Myoglobin : 24% Identity
• Hemoglobin subunits and myoglobins share an evolutionarily conserved
structural pattern called “globin fold”.
Oxygen Binding Properties of Myoglobin and Hemoglobin

Mb : Hyperbolic
P50 = 2 torr Hb : Sigmoidal
P50 = 26 torr

• 2,3-bisphosphoglycerate in red blood cell significantly lowers oxygen

binding affinity of hemoglobin.
• 4 independent oxygen bindings
• Cooperative bindings:
Physiological significance of cooperative binding

1. Cooperative binding leads to efficient oxygen transport.

lung (~100 torr) vs tissue (~20 torr)

2. Cooperative release of oxygen favors more complete delivery to

tissues.

3. Cooperative binding leads to efficient oxygen transport.

 Loading & Unloading of Oxygen by Hemoglobin

X10 than myoglobin, X1.7 than noncooperative protein

• O2 Uptake by Hemoglobin in Lung (pO2 ~100 torr; 98% occupancy)

• O2 Release in Typical Peripheral Tissues (pO2 ~20 torr; 32% occupancy; 66% Release)
• O2 Unloading in Resting Muscle (pO2 ~40 torr; 77% occupancy; 21% Release)
• O2 Unloading in Exercising Muscle (pO2 ~20 torr; 32% occupancy; 67% Release)
O2 Binding Changes the Quaternary Structure of Hemoglobin

• 15 degree rotation of a1b1 dimer and a2b2 dimer upon oxygen binding
• Overall structure of dimer themselves is relatively unchanged.
• The interface between a1b1 dimer and a2b2 dimer is significantly changed.
• Deoxyhemoglobin : T (for Tense) State; Stronger Inter-Subunit Interactions
• Oxyhemoglobin : R (for Relaxed) State; Weaker Inter-Subunit Interactions
• In R state, oxygen binding sites are free of strain and show higher affinity
 Hemoglobin Cooperativity (1)
Concerted Model (MWC Model)

• Oxygen Binding Affinity : T state < R state

• Oxygen binding shifts the quaternary structure of hemoglobin from T to R.
• Oxyhemoglobin favors R state, whereas deoxyhemoglobin is in T state.
• This model postulates that all 4 subunits are in the same conformation.
 Hemoglobin Cooperativity (2)
Sequential Model (allosteric)

• Oxygen Binding Affinity : square state ( ) < quarter circle state ( )

• Oxygen binding modulates the tertiary structure of the neighboring subunits.
• This model postulates that the individual subunits can have different conformations.
 Hemoglobin Cooperativity (3)
Which one is Truth ?

• Neither the concerted model nor the sequential model is fully accurate.

• Oxygen Binding Affinity  Hemoglobin-[O2]0 (T) : Hemoglobin-[O2]3 (R) = 1 : 20

(Concerted Model Works !!!)

• Oxygen Binding Affinity  Hemoglobin-[O2]0 (T) : Hemoglobin-[O2]1 (T) = 1 : 3

(Sequential Model Works !!!)

Structurally, [O2]3 (T)= [O2]4 (R)

Affinity-wise, [O2]0 = [O2]1
 Oxygen Binding to Heme Changes Interface Structures

• Oxygen binding causes T to R quaternary structure changes

• Oxygen binding induces heme plane movement.
 Heme plane movement prompts conformational changes of hemoglobin subunit via
proximal histidine residue.
 The C-terminal end of the individual subunit, which is located in the inter-subunit space, is
relocated upon oxygen binding.
 The structural transition at the iron in one subunit is directly transmitted to the other
subunits changing the interface between the ab dimers.
 Cooperative oxygen binding by hemoglobin
The movement of the proximal histidine and its associated
α helix upon oxygen binding drives cooperativity.
The oxygen-binding curves for pure hemoglobin and
hemoglobin in red blood are markedly different.
 Specific Binding of 2,3-BPG to T-State Hemoglobin

Allosteric effector
Bind in sites other than
the active site

• The T state of hemoglobin is highly unstable. (thermodynamically not favorable.)

• During T  R transition, the pocket collapses, 2, 3-BPG is released.
• But, R  T transition is essential for efficient oxygen unloading.
• In red blood cell, [Hb] = [2,3,-BPG] = ~2 mM.
• 2,3-BPG can bind to the central pocket located in the center of the tetramer.
• 2,3-BPG binding pocket can be generated only in the T-state.
• Thus, 2,3-BPG can stabilize the T form population.
 Cargo Transport Efficiency of Hemoglobin vs. 2,3-BPG

• [2,3-BPG]LOW  R > T and [2,3-BPG]HIGH  R < T (Allosteric Regulation)

• O2 unloading efficacy  2,3-BPG(-) : 2,3-BPG(+) = 8% : 66%
• Adult Hemoglobin [ab]2 vs. Fetal Hemoglobin [ag]2
• In g chain, serine residue has replaced 143 Histidine residue of b chain, which
provides a positive charge critical for optimum 2,3-BPG binding.
• Thus, 2,3-BPG binding is much less efficient for fetal hemoglobin.
• Oxygen affinity : fetal hemoglobin > maternal hemoglobin.
 The Bohr Effect (1) : Hydrogen Ion (pH)

• Rapidly metabolizing tissues such as contracting muscle generate large amounts of

hydrogen ions.
• Decrease of pH from 7.4 to 7.2 increases oxygen unloading efficiency by 11%.
• Salt bridges involving Lys40 of a2, His146 of b1 and Asp94 of b1 stabilize the T form
of hemoglobin.
• Protonation on His146 of b1 enhances salt bridge formation with Asp94 of b1.
The Bohr Effect (2) : Carbon Dioxide (1)

Constant pH!

• Actively metabolizing tissues release large amounts of CO2 resulting in pH decrease.

• Carbonic Anhydrase : Carbon Dioxide  Carbonic Acid (pKa 3.5)  Bicarbonate + H+

• 40 torr CO2 increases oxygen unloading efficiency by 11% at pH 7.2.

CO2 decrease the affinity of hemoglobin for oxygen beyond the effect due to decrease in pH
 The Bohr Effect (3) : Carbon Dioxide (2)

• CO2 can react with the N-terminal amino group of the hemoglobin subunits and

yield carbamate groups, which produce additional negative charges.

• The amino termini reside in the interface of ab dimers.

• These CO2-induced negative charges in the N-termini of hemoglobin subunits

consequently promote salt bridges formation stabilizing the T form.

Carbon monoxide is rapidly transferred
from carboxy-hemoglobin to Ngb-H64Q
 Sickle Cell Anemia

Sickle-Shaped RBC Deoxyhemoglobin S Hemoglobin Fibers

@ oxygen low
• In Sickle-Cell Anemia, Glu6 of b chain is mutated to Val6 (HbS substitution).
• The deoxy HbS b chains can establish aberrant hydrophobic interactions involving
Val6 of one b chain and Phe85 and Val88 of another b chain (i.e. between different
hemoglobin tetramers).  Deoxyhemoglobin polymerization  Formation of
hemoglobin fibers (i.e. insoluble aggregates of hemoglobin)
• The HbS substitution does not alter the properties of oxyhemoglobin because the
hydrophobic interaction between Val6 and Phe85/Val88 is not allowed in R state.
3.4 The Immune System Depends on Key Binding
Proteins

The immune systems address the problem of recognition of

non-self in the context of self
 Recognition of Toll-like receptors (TLRs)

1. Each LRR typically contains 20–30 residues,

including 6 that are usually leucine

2. human TLRs have from 18 to 27 LRR

repeats

3. Each TLR targets a specific molecular class,

often called a pathogen-associated
molecular pattern (PAMP), found primarily
on invading organisms.

4. Typically, a PAMP is a critical and

widespread component of the pathogen.

leucine-rich repeats (LRRs)

 How do TLRs recognize PAMPs?
PAMPs for TLR3 are dsRNA molecules of appropriate lengths fitting in the cavity
between two monomers

1. The TLR3 recognizes its PAMP, a dsRNA.

2. The structure of the complex between two molecules of the extracellular
domain of TLR3 reveals a 46-base-pair dsRNA can be bound tightly.
3. [dsRNA] < more than 1000 times [dsDNA]
Antibodies (immunoglobulins) bind specific molecules
through specific hypervariable loops.

Even those that the adaptive immune has never encountered in the
course of evolution, antibodies produced function to recognize foreign
molecules and mark them, signaling foreign invasion.
 Sequence variability

Immunoglobulin sequences include regions of extensive sequence variability.

For portions of these IgG molecules, especially 3 stretches of approximately

7 to 12 amino acids within each chain, these sequences are highly variable.
These segments are referred to as the hypervariable regions.
 First, three loops at one end of the structure that contain
hypervariable regions form a potential binding surface.
These loops are referred to as hypervariable loops or
complementarity-determining regions (CDRs).

Second, the amino terminus and the carboxyl terminus

are at opposite ends of the structure, which allows
structural domains to be strung together to form
chains.

The immunoglobulin fold consists of a pair of β sheets

linked by a disulfide bond. Hydrophobic interactions occur
with three hypervariable loops at one end of the structure.
 Hypervariable
loops

The 12 immunoglobulin fold domains in an IgG molecule are arranged

to form a Y-shaped structure, comprising two identical arms with
hypervariable regions at the end.
Immunoglobulin G molecules can be
cleaved into two fragments and one
fragment by treatment with papain.

Fab is the antigen binding site whereas

Fc is a structural and functional domain.
 Fab

1. The structure of an fragment reveals how the six hypervariable loops come
together to form a binding surface.

2. All six CDRs of the antibody participate directly in molecular recognition.

3. The region of contact is quite extensive (about 30 X 20 Å ), and the apposed

surfaces are rather flat.
 Binding between lysozyme and a specific fragment.

1. The side chain of glutamine 121 of lysozyme penetrates deeply into the antibody’s binding site, where it
forms a hydrogen bond with a main-chain carbonyl oxygen atom and is surrounded by 3 aromatic side
chains.

2. The formation of 12 hydrogen bonds and numerous van der Waals interactions contributes to the high
affinity (L½ = 20 nM) of this antibody–antigen interaction.

In conclusion, many hydrogen bonds, ionic interactions, and van der Waals interactions, reinforced by
hydrophobic interactions, combine to give specific and strong binding.
Five classes of immunoglobulin have the same light chain combined with a
different heavy chain (γ​, α, μ​, δ, or ε). The light chains are shown in yellow,
and disulfide bonds are indicated by green lines.

IgM has a noteworthy pentameric structure with ten potential antigen-binding sites. IgMs
are the first class of antibody produced after an infection
Recombination events equip the adaptive immune
system with millions of unique antibodies

Immunoglobulin gene undergoes recombination events that introduce different combinations of CDRs,
resulting in more than 300 possible light chains and more than 8000 possible IgG heavy chains.

As each light chain can come together with each heavy chain, this produces more than 300 X 8000 =
2,400,000 potential antibodies.

This explains how the adaptive immune system can respond to unknown invaders; it is prepared with
many potential molecular recognition templates, some of which will adequately match any new threat.
Antibodies elicited against the spike protein of SARS-CoV-2 can bind
to this protein and block the ability of the virus to infect cells.

The vaccines can induce antibodies that bind

to the spike protein and block its ability to bind
to and infect cells.
The structure of the extracellular domain In class I MHC proteins, peptides can bind in the groove. The
of a class I MHC protein reveals a deep peptide in red binds in a relatively extended conformation.
groove. The domain is a complex
between 2 different polypeptide chains,
shown in blue and yellow.
 Some recurring features of Peptide in MHC class I protein

(A) The amino acid sequences of 3 peptides that bind to the class I MHC protein HLA-A2. Each of these
peptides has leucine in the second position and valine in the carboxyl-terminal position.

(B) Peptides always have leucine in the second position and valine in the last position (Figure 3.47). Side
chains from the MHC molecule interact with the amino and carboxyl termini and with amino acids in
these two key positions. These two residues are often referred to as the anchor residues.
The T-cell receptor recognizes specific class I MHC complexes,
contacting both the MHC protein and the bound peptide.

1. These T-cell receptors are closely related to the Fab portion of an

antibody.

2. Each T-cell receptor is capable of recognizing an MHC-peptide

complex by forming a structure analogous to an antibody–antigen
complex (Top to the peptide).

3. This process can fail, as revealed by the occurrence of autoimmune

diseases, such as type 1 diabetes, rheumatoid arthritis, and dozens of
other conditions.
3.5 Quantitative Terms Can Describe Binding Propensity

The tendency of a receptor to bind a particular ligand depends on

the ligand concentration.
1. Binding can be monitored by measuring the fraction of receptors with ligand bound as a
function of the amount of ligand added.

2. The data curve is characterized by the value of Kd, which is the same as L1/2 .
At high enough concentrations, ligands with lower affinity for a receptor can compete for binding
with high-affinity ligands.

At a BPA concentration of 1 μM, the fraction of estrogen receptor bound to estradiol is reduced
from 0.5 to 0.14.
At equilibrium, the rates of the forward and reverse reactions are the same.
In (A), no ligands are bound and the forward reaction will proceed faster than the reverse reaction to
reach the equilibrium distribution in (B). In (C), all receptor sites are filled and the reverse reaction will
proceed faster to reach (B). At equilibrium, the forward and reverse reactions can proceed at the
same rates to interconvert the distributions in (B) and (D), which have the same fraction of receptors
filled.
A rate constant reflects the distribution of reaction times at the single molecule level.

The results of a simulation with 1000 events of dissociation times for an estrogen
receptor–tamoxifen complex are shown, with dissociation times ranging from nearly zero
to more than 50 minutes, with an average of 12 minutes.

The rate constant of 1.0 X 10-3 S-1 characterizes the exponential decline in this distribution,
shown in red.