MOLECULAR BIOTECHNOLOGY

Design and Synthesis of Bioactive Compounds

Luigi Vitagliano

Istituto di Biostrutture e BioimmaginiConsiglio Nazionale delle RicercheVia Mezzocannone 16, 80134 Napoli

MOLECULAR BIOTECHNOLOGY

Design and Synthesis of Bioactive Compounds

Lecturer : Luigi Vitagliano

Phone: 0812534506

E-mail: luigi.vitagliano@cnr.it

Slides of the lectures can be downloaded from the page

Additional material for further reading will be provided

throughout the course

Synthetic program of the course

Structural Biology

The state of the art

Experimental approaches

X-ray crystallography

Electron microscopy

Computational approaches

Protein structure prediction methods

Molecular dynamics simulation

Structure validation

Successful examples: The ribosome machinery and the DNA structure

Rational Design of bioactive molecules

Properties of protein structures

Ligand-protein interactions

Structure-based drug discovery

Ligand-based drug discovery

Fragment-based drug discoveryClassical and recent successful examples of rational drug design

Lesson 1

Part a

Structural Biology

• The state of the art

• Experimental approaches

• X-ray crystallography

Overview of the Structure-based drug discovery

A key factor in the success of this strategy is the accurate structural

characterization of the target that is frequently a protein or a nucleic acid

Structure-based drug designi

Biomolecoles

‘Small’ molecoles: Water, Vitamines, Hormons, Lipids, Metabolites…

Macromolecules: Proteins, Nuclecic acids (DNA/RNA), Polysaccharides

Molecules found in living organisms

HemoglobinC2952H4664O832N812S8Fe4

Proteins are characterized by a remarkable structural complexity being made of thousands of atoms

WaterH2O

GlucoseC6H12O6

Complexity of Nucleic acids: DNA and RNAChains of DNA are made of millions of nucleotides

Current record for sequence deconding is of 2.3 million bases of human DNA in one step

Formula Cxxxxxxxx Oxxxxxxx Hxxxxxxxx Pxxxxxx Nxxxxxxxx

Chimie dans l’espace - Discovery and importance of

stereochemistry (1875)

The definition of the three-

dimensional structure is crucial for

macromolecules since they may

adopt, in principle, an enormous

number of distinct states

Structure – function relationships in water

H2O

The central oxygen atom binds two hydrogen atoms

and has two lone pairs of electrons

VESPR Rules

Bent structure

Negative pole

Positive pole

Structure – function relationships in water

+

- +

+ -

-

+

+ - +

+ --

+ - +

+ ---

-

Ionic crystal

+

-

Hydration

A biochemical/biological problem

Identification of the key molecular players

Preparation of the samples for the structural investigation

Experiment(s)

Analysis of the biomolecule structure

If everything works, you end up with ……

…..many additional biochemical/biological problems

The structural biological approach

Protein Chemistry

Linear polypeptide chain

N Nitrogen

O Oxygen

C Carbon

H Hydrogen

R Side Chain

Protein three-dimensional structure

Structure - Function relationship

Fibrous proteins These proteins are characterized by very repetitive sequence motif (examples Collagen, wool,

silk…) - They have frequently a structural (mechanical) role – They share significant analogies

with synthetic polymers.

Globular proteins These are characterized by a rough globular shape and presents a large variability in

aminoacid composition and sequence.

Intrinsically disordered proteins (IDP)These are proteins that do not present an intrinsic well-defined three-dimensional structure in

their biological active state. They tend to become structured upon interaction with other

biological partners. They often have a low complexity sequence (repetitions of specific

aminoacid traits).

All type of proteins play crucial role in biological processes. From a chronological point of view

fibrous proteins have been the first to be structurally characterized. They were followed by

globular proteins, that for their huge diversity and complexity are far to be fully characterized.

IDPs represent an emerging subject of the scientific community.

Proteins are often classified according to their structural properties

The first structural characterization of a globular protein was accomplished by

Kendrew (Myoglobin 1958)

The aspect of Kendrew’s model of myoglobin was a horrible, visceral-looking

object, since he had used a long sausage of plasticine

What about the structure of globular proteins?

Upon seeing the structure of myoglobin (Fig. 1) at 6 Å resolution, Kendrew et al. said, “Perhaps the most remarkable features of the molecule are its complexity and its lack of symmetry. The arrangement seems to be almost totally lacking in the kind of regularities which one instinctively anticipates, and it is more complicated than has been predicated by any theory of protein structure. Though the detailed principles of construction do not yet emerge, we may hope that they will do so at a later stage of the analysis.”

Enchanting protein shapes of globular proteins

90°

Scaring protein shapes

Resuscitation promoting factor B da M. tuberculosis

.

A classical exampleHemoglobin Sickle Cell Anaemia

This is genetically tramistted since in heterozygotes the mutation producese resistenceagainst malaria The mutated gene is wdespreadin Africa

Due to its high concentration mutated Hb may form fibers

A single residue mutation may cause a disease

Mutation : Glu Val

In erythrocytesHemoglobin isabundant: 340 mg/ml

Deoxy form

Normal and mutated Hb

Fructose intoleranceMutations in Aldolase B

Incidence 1/20000.

It is fundamental the elimination of fructose from diet

A single residue mutation may cause a disease

Misfolded proteins

Globular proteins in specific conditions may aggregate to form insoluble species Amyloid fibers

Beta-rich structures

Misfolding of proteins ‘

Trasmissible encelopathiesCow

Human Disease: vCJD variant of the diseaseCreutzfeldt-Jakob (nota dal 1996)

Determination of the three-dimensional structure of

biological macromolecules

The status of the art

1 Å 1 nm 10 nm 1 mm 10 mm 0.1 mm100 nm

NMR

X-ray crystallography

AFM

single particle Cryo-EM

Electron tomography

Light microscopy

atoms proteins viruses bacteria cells

Techniques generally used for the structural

characterization of Bio-macromolecules

The paradigm of function-structure relationships

Structure - Function

Experiments

X – Ray crystallography

NMR Nuclear Magnetic resonance

Cryo-EM Electron Microscopy

‘Modeling’

Molecular dynamics

‘Docking’

Computations

Number of protein structures reported in the

Protein Data Bank

The vast majority of these structures (85%) has been so far

determined by X-ray crystallography

Some classical triumps of x-ray crystallography

a) Ribosome

b) Rhodopsin

c) Reovirus core particle

d) HMG-CoA reductase

e) RNA polymerase II

f) ATP synthase

g) Nucleosome core particle

h) Mismatch repairprotein

g) HIV envelope glycoprotein

gp120

Not only large macromolecules……..

Examples of recent fundamental peptide structural studies

Amyloid-forming peptides (4 to 7 residues long)These structures became models for fibers of proteins involved in neurodegenerative diseases

X-ray structures

David Eisenberg group

Nelson et al. 2005, Nature

Sawaya et al 2007 Nature

Wiltzius et al. 2009 Nature Structure and Molecular biology

Sawaya et al. 2009 PNAS

Apostol et al. 2010 JBC

X-ray crystallography and proteins: a happy marriageSince 1901 26 Nobel prizes have been awarded to 43 sceintists in disciplines

related to crystallography. A significant portion of these were for the characterization of large biomolecules

Recent Nobel prizes awarded to studies conducted using X-ray diffraction

2012 Chemistry

Lefkowitz e Kobilka –G-coupled receptors- Protein Crystallography

2011 Chemistry

Shechtman – Discovery of quasicristalls– Basic crystallography

2010 Physics

Geim e Novoselov – Graphene discovery and characterization – Materials crystallography

2009 Chemistry

Ramakrishnan, Steitz e Yonath – Ribosome structure– Protein Crystallography

2006 Chemistry

Kornberg – Tracription factors– Protein Crystallography

2003 Chemistry

MacKinnon – Potassium channels– Protein Crystallography

2017 CrioElectron Microscopy, 2013 Computational biology - 2002 NMR of proteins

This scenario is going to change soon….The coming era of cryo-electron microscopy

After fifty years (and thousands of publications)

the mecahnism is still debated, Sept 2013

Protein funtionality: not just structure

Importance of protein dynamics

How does haemoglobin work?

Determination of the three-dimensional structure of

biological macromolecules by X-ray diffraction

techniques

Basic experiment

Main components

X-ray

Sample

Detector

Proprieties of the X-rays

Electromagnetic radiation with wavelength

between 0.1 and 1000 Å

Conventional X-ray generator

X-ray sources for structural studies on proteins

Conventional

Synchrotron

Syncrothron are devices in which charged particles (electron or

positron) circulate with speed close to that of light

The acceleration of these particles generates radiations

Components of a synchrotron

Bending magnets

Focus

Diffraction

Image

Scheme of diffraction and imaging

Object Object

Detector

Lens

Diffraction

Diffraction

pattern

Diffraction

Single emitter

Constructive

interference

Destructive

interference

Diffraction & Interference

Diffusion from a multiple source

Diffusion from disordered scatters

Diffusion from ordered scatters

A diffraction image

Take home messages

Scatters in these experiments are electrons – therefore we get

direct information on them

Sharp diffraction is a result of the ordered organization of the

electrons in the sample

All electrons of our sample contribute to the scattering in each

direction – each spot on the diffraction diagram depends on the

location of all electrons

Crystallography versus Spectroscopy

Single crystal diffraction For systems with well defined shapes that could generate ordered

crystals - organic molecules, oligonucleotides and globular proteins

Fiber diffraction analysesFibrous systems

X-ray diffraction analyses

Three-dimensional crystals

A diffraction image

In fibers the molecular entities are ordered along the so-called

fiber axis

Fiber X-ray diffraction analysis

Theory

Fiber diffraction

The discovery of the alpha-helix (1951)

The fiber diffraction

pattern

The 3D model

by Pauling and Corey

Wool diffraction pattern:

meridional reflection (5.1 Å)

equatorial reflection (10 Å)

Successful modelling of other biomacromolecules came using

the same approach

Beta-sheet Pauling and Corey, 1951

Collagen Ramachandran and

Triple Helix Kartha 1954

Rich and Crick, 1961

The most famous achievement of X-ray fiber diffraction

analysis

The DNA structure

analisys

Single Crystal X-ray diffraction analysis

Data Collection

Data Analysis

PROTEIN

CRYSTALLOGRAPHY

Main steps

Single crystal X-ray crystallography:

The experimental setup

Example of protein crystals

X-ray sources for protein crystallography

Synchrotron

Conventional

Crystallization

Phase transition: liquid - solid

Aqueous solution

of a protein

Amorphous solid

Crystal

Isotropic solid characterized by an

irregular organization of the

atoms/molecules (for example glass)

Homogeneous and anisotropic solid

characterized by a regular organization

of the atoms/molecules in the space

Crystals are characterized by the repetition in the

three-dimensional space of a specific volume

that is defined as unit cell

a

b

c

Unit cell

The repeating structural motif may be:

an ion, an atom, a molecule or an ensemble

of molecules.

Three-dimensional lattice

The seven crystal classes

The unit cell is defined by three

axes a, b, c and three angles.

Different combinations are

possible

Examples of protein crystals

Due to their internal symmetry crystal are solids with planar

faces as polyhedra

Main features of protein crystals

• High solvent (water) content (20-80%)

• Large solvent channels – the interactions between

mates in the crystals are weak

• Limited level of order – Limited size of protein crystals

• High fragility and sensitioty to external conditions

[Linear dimensions 0.1-1.0 mm (= 1015-1020 molecules);

Inorganic crystals may be seen at macroscopic level (for example quarz)]

[Salt bridges, hydrogen bonds, van der Waals interactions – a small fraction

of the protein atoms are involved in packing contacts]

[pH variations, temperature, ionic strength…]

Crystal packing:

Elongation factor G

Solvent channels

• the protein may

be active

• it is possible to

diffuse small

molecules in the

crystals as ligands

and inhibitors

(soaking)

Ca trace

Needs for crystallization

• Purity = absence of contaminants

• Homogeneity = absence of conformational (inter-

domain flexibility, equilibrium between conformers, aggregation, partial

denaturation…) and/or sequence (fragmentation, uncomplete

post-translational modifications… ) heterogeneity

• Quantity

• Biological activity

• Purity

Effects of contaminants:

They may prevent crystal formation

They may perturb the crystal growth

They may favor the growth of disordered crystals

SDS gel: occurrence and

oimension of peptide/protein

contaminants

Needs for crystallization

How can crystallization may be induced?

Phase transition: liquid - solid

Solubility (cs): maximum solute concentration at a given

temperature

Sovrasaturation (cp/cs) non-equilibrium; the concentration

of the solute (cp) is larger than its solubility (cs)

solubility

Solubility curve = solid-liquid

equilibrium

Co

nc. p

rote

in

Conc. precipitant

(A parameter that decreases

the protein solubility:

temperatura, pH, salt, …)

Phase Diagramprecipitation

sovrasaturation

1) Nucleation: formation of stable aggragates high

sovrasaturation

2) Growth It may occur in the nucleation zone but is more

favore in the metastable region

3) End of the growth

Studies carried out on lisozyme

indicate that critical aggregates

are made of 30-40 molecules.

Main chemico-physical parameters that affect

protein solubility

• Protein concentration

• Concentration and nature of the precipitating agent :

Salts: protein dehydration

Organic solvent : for example alcohols may reduce the

dielectric constant

Organic polymers (PEG, poliamynes)

• Temperature

• pH of the buffer

• Ionic strength

Vapour diffusion techniques(Hanging - Sitting Drop)

Closed system: [ drop of (protein + precipitants) + precipitants+ air] it

evolves toward the equilibrium through the evaporation of the water

from the drop to the reservoir

Hanging Sitting

Examples of experimental settings

Soluzione

madre

Factors that (may) affect protein crystallization

• Nature and concentration of the precipitant

• Protein concentration

• Buffer and pH

• Temperature

• Ionic strength

• Purity and homogeneity of the sample

• Additives, effettors, ligands

• Source of the macromolecule (organism, recom.)

• Redox status of the environement

• Ions

• Detergents

• Gravity, convection, sedimentation

Protein crystallization faclities

RobotsCrystallization under microgravity

conditions

“Beuty is not enough”

What is important is the diffraction pattern!

A real diffraction image

A more real case – resolution is limited

Diffraction spot magnification

Intensities of the pixels that constitute the spot

Integration of the spot intensity

Measure of the spot intensities

These intensities are proportional to the square of the

module of the structure factore F(hkl)

The structure factor is linked to the electron density

through an operation called Fourier

h k l

lzkykxi2explkhFV

1zvxρ

Protein crystallography: basic concepts

Electron density

map

model

fitting

Model building in the elctron density maps

Main chain

Side chains

Risolution structural details

3.5Å 4Å

bassa risoluzione

6Å: Solo caratteristiche grossolane

del modello sono rintracciabili. Ad

es. a-eliche.

3Å: Polypeptide chain trace

2Å: Backbone conformation

1.5Å : Individual atoms in the maps.

Detailed description of the solven

< 1.2Å: Hydrogen atoms (may)

become visible. 1.0Å altissima risol. 2.5Å media risol.

Atomic resolution: better than 1.2 Å

“ultra-high resolution”: RNase A 0.87 Å

Individual atoms

Double conformations

Hydrogen atoms

JMB, 297, 713-732, 2000

ATOM 1 N VAL A 1 10.720 19.523 6.163 1.00 21.36

ATOM 2 CA VAL A 1 10.228 20.761 6.807 1.00 24.26

ATOM 3 C VAL A 1 8.705 20.714 6.878 1.00 18.62

ATOM 5 CB VAL A 1 10.602 22.000 5.966 1.00 27.19

ATOM 6 CG1 VAL A 1 10.307 23.296 6.700 1.00 31.86

ATOM 7 CG2 VAL A 1 12.065 21.951 5.544 1.00 31.74

ATOM 8 N LEU A 2 8.091 21.453 7.775 1.00 16.19

ATOM 9 CA LEU A 2 6.624 21.451 7.763 1.00 17.31

ATOM 10 C LEU A 2 6.176 22.578 6.821 1.00 18.55

ATOM 11 O LEU A 2 6.567 23.730 7.022 1.00 18.72

ATOM 12 CB LEU A 2 6.020 21.707 9.129 1.00 18.34

ATOM 13 CG LEU A 2 6.386 20.649 10.198 1.00 17.39

ATOM 14 CD1 LEU A 2 5.998 21.119 11.577 1.00 17.99

ATOM 15 CD2 LEU A 2 5.730 19.337 9.795 1.00 16.96

ATOM 16 N SER A 3 5.380 22.237 5.852 1.00 15.02

ATOM 17 CA SER A 3 4.831 23.237 4.928 1.00 16.59

ATOM 18 C SER A 3 3.725 24.027 5.568 1.00 14.84

ATOM 19 O SER A 3 3.095 23.717 6.591 1.00 14.40…

X Y Z

Coordinates Occup. B-factorAtom

Residue

A typical PDB file