today’s lecture

Experimentally solving protein structures and protein-protein

interactions

Lecture 21

Introduction to Bioinformatics2007

CENTR

FORINTEGRATIVE

BIOINFORMATICSVU

E

Today’s lecture

1. Experimental techniques for determining protein tertiary structure

2. Protein interaction and dockingi. Ribosome example

ii. Zdock method

3. Molecular motion simulated by molecular mechanics

If you throw up a stone, it is Physics.

If you throw up a stone, it is Physics. If it lands on your head, it is Biophysics.


If you write a computer program, it is Informatics.


If you write a computer program, it is Informatics. If there is a bug in it, it is Bioinformatics

Experimentally solving protein structures

Two basic techniques:

1. X-ray crystallography

2. Nuclear Magnetic Resonance (NMR) tchniques

1. X-ray crystallography

Purified protein

Crystal

X-ray Diffraction

Electron density

3D structureBiological interpretation

Crystallization

Phase problem

Protein crystals• Regular arrays of protein molecules

• ‘Wet’: 20-80% solvent• Few crystal contacts

• Protein crystals contain active protein• Enzyme turnover• Ligand binding

Example of crystal packing

Examples of crystal packing

2 Glycoprotein I~90% solvent (extremely high!)

Acetylcholinesterase~68% solvent

Problematic proteins (no crystallisation)

• Multiple domains

• Similarly, floppy ends may hamper crystallization: change construct

• Membrane proteins

• Glycoproteins

Flexible

Lipid bilayer

hydrophilic

hydrophilic

hydrophobic

Flexible and heterogeneous!!

Experimental set-up• Options for wavelength:

– monochromatic, polychromatic – variable wavelength

Liq.N2 gas stream

X-ray source

detector

goniometer

beam stop

Diffraction imageDiffraction image

Water ring

Diffuse scattering (from the fibre loop)

reciprocal lattice reciprocal lattice (this case hexagonal)(this case hexagonal)

Beam stop

Increasing resolution

Direct beam

ReflectionsReflections ( (h,k,lh,k,l) ) withwith I( I(h,k,lh,k,l))

The rules for diffraction: Bragg’s law

• Scattered X-rays reinforce each other only when Bragg’s law holds:

Bragg’s law: 2dhkl sin = n

Phase Problem

• Determining the structure of a molecule in a crystalline sample requires knowing both the amplitude and the phase of the photon wave being diffracted from the sample

• X-rays which are emitted start out with dispersed phases, and so the phases get lost

• Unfortunately, phases contribute more to the informational content of a X-ray diffraction pattern than do amplitudes. It is common to refer to phaseless X-ray data as having "lost phases“

• Luckily, several ways to recover the lost phases have been developed

Building a protein model• Find structural elements:

-helices, -strands• Fit amino-acid sequence

Effects of resolution on electron density

Note: map calculated with perfect phases

d = 4 Å

d = 3 Å



d = 2 Å



d = 1 Å



Refinement process

• Bad phases poor electron density map

errors in the protein model

• Interpretation of the electron density map improved model

improved phases improved map

even better model

… iterative process of refinement

Validation

• Free R-factor (cross validation)– Number of parameters/

observations• Ramachandran plot • Chemically likely (WhatCheck)

– Hydrophobic inside, hydrophilic outside

– Binding sites of ligands, metals, ions

– Hydrogen-bonds satisfied– Chemistry in order

• Final B-factor (temperature) values

2. Nuclear Magnetic Resonance (NMR)

800 MHz NMR spectrometer

Nuclear Magnetic Resonance (NMR)

• Pioneered by Richard R. Ernst, who won a Nobel Prize in chemistry in 1991, FT-NMR works by irradiating the sample, held in a static external magnetic field, with a short square pulse of radio-frequency energy containing all the frequencies in a given range of interest.

• The polarized magnets of the nuclei begin to spin together, creating a radio frequency (RF) that is observable. Because the signals decays over time, this time-dependent pattern can be converted into a frequency-dependent pattern of nuclear resonances using a mathematical function known as a Fourier transformation, revealing the nuclear magnetic resonance spectrum.

• The use of pulses of different shapes, frequencies and durations in specifically-designed patterns or pulse sequences allows the spectroscopist to extract many different types of information about the molecule.

Nuclear Magnetic Resonance (NMR)• Time intervals between pulses allow—among other things—magnetization

transfer between nuclei and, therefore, the detection of the kinds of nuclear-nuclear interactions that allowed for the magnetization transfer.

• Interactions that can be detected are usually classified into two kinds. There are through-bond interactions and through-space interactions. The latter usually being a consequence of the so-called nuclear Overhauser effect (NOE). Experiments of the nuclear-Overhauser variety may establish distances between atoms.

• These distances are subjected to a technique called Distance Geometry which normally results in an ensemble of possible structures that are all relatively consistent with the observed distance restraints (NOEs).

• Richard Ernst and Kurt Wüthrich —in addition to many others— developed 2-dimensional and multidimensional FT-NMR into a powerful technique for the determination of the structure of biopolymers such as proteins or even small nucleic acids.

• This is used in protein nuclear magnetic resonance spectroscopy. Wüthrich shared the 2002 Nobel Prize in Chemistry for this work.

Gly

Gly

AspAsn

Asp

Phe

ThrSer

Leu

Val

2D NOESY spectrum

• Peptide sequence (N-terminal NH not observed)• Arg-Gly-Asp-Val-Asn-Ser-Leu-Phe-Asp-Thr-Gly

NMR structure determination: hen lysozyme

• 129 residues– ~1000 heavy atoms– ~800 protons

• NMR data set– 1632 distance restraints– 110 torsion restraints– 60 H-bond restraints

• 80 structures calculated• 30 low energy

structures used 0

2000

4000

6000

8000

1 10 4

1.2 10 4

10 20 30 40 50 60 70

Tot

al e

nerg

y

Structure number

Solution Structure Ensemble

• Disorder in NMR ensemble– lack of data ?– or protein dynamics ?

Problems with NMR

• Protein concentration in sample needs to be high (multimilligram samples)

• Restricted to smaller sized proteins (although magnets get stronger)

• Uncertainties in NOEs introduced by internal motions in molecules (preceding slide)

X-ray and NMRsummary

• Are experimental techniques to solve protein structures (although they both need a lot of computation)

• Nowadays typically contain many refinement and energy-minimisation steps to optimise the structure (next topic)

X-ray and NMRsummary (Cntd.)

• X-ray diffraction– From crystallised protein sample to electron

density map• Structure descriptors: resolution, R-factor, B-factor

• Nuclear magnetic resonance (NMR)– Based on atomic nuclear spin – Produces set of distances between residues

(distance restraints)– Distances are used to build protein model using

Distance Geometry (a technique to build a protein structure using a set of inter-residue distances)

Protein binding and protein-protein interactions

• Complexity:– Multibody interaction

• Diversity:– Various interaction types

• Specificity:– Complementarity in shape and binding

properties

Protein-protein interactions• Many proteins interact through

hydrophobic patches

• Hydrophobic patches often have a hydrophilic rim

• The patch-rim combination is believed to be important in providing binding specificity

hydrophobic

very hydrophilic

hydrophilic

PPI Characteristics• Universal

– Cell functionality based on protein-protein interactions• Cyto-skeleton• Ribosome• RNA polymerase

• Numerous– Yeast:

• ~6.000 proteins• at least 3 interactions each~18.000 interactions

– Human:• estimated ~100.000 interactions

• Network– simplest: homodimer (two)– common: hetero-oligomer (more)– holistic: protein network (all)

Interface Area• Contact area

– usually >1100 Å2

– each partner >550 Å2

• each partner loses ~800 Å2 of solvent accessible surface area– ~20 amino acids lose ~40 Å2

– ~100-200 J per Å2

• Average buried accessible surface area:– 12% for dimers– 17% for trimers– 21% for tetramers

• 83-84% of all interfaces are flat• Secondary structure:

– 50% -helix– 20% -sheet– 20% coil– 10% mixed

• Less hydrophobic than core, more hydrophobic than exterior

Complexation Reaction

• A + B AB

– Ka = [AB]/[A]•[B] association

– Kd = [A]•[B]/[AB] dissociation

Experimental Methods for determining PPI• 2D (poly-acrylamide) gel electrophoresis mass spectrometry• Liquid chromatography

– e.g. gel permeation chromatography• Binding study with one immobilized partner

– e.g. surface plasmon resonance• In vivo by two-hybrid systems or FRET• Binding constants by ultra-centrifugation, micro-calorimetry or

competition• Experiments with labelled ligand

– e.g. fluorescence, radioactivity• Role of individual amino acids by site directed mutagenesis• Structural studies

– e.g. NMR or X-ray

PPI Network

http://www.phy.auckland.ac.nz/staff/prw/biocomplexity/protein_network.htm

Binding vs. Localization

Obligateoligomers

Non-obligateweak transient

Non-obligatetriggered transient

e.g. GTP•PO4-

Non-obligateco-localised

e.g. in membrane

Non-obligatepermanent

e.g. antibody-antigen

strong

weak

co-expressedand at same place

different places

Some terminology

• Transient interactions:– Associate and dissociate in vivo

• Weak transient:– dynamic oligomeric equilibrium

• Strong transient:– require a molecular trigger to shift the equilibrium

• Obligate PPI:– protomers no stable structures on their own (i.e. they

need to interact in complexes)– (functionally obligate)

Analysis of 122 Homodimers

• 70 interfaces single patched

• 35 have two patches

• 17 have three or more

Interfaces

• ~30% polar

• ~70% non-polar

Interface• Rim is water accessible

riminterface

Interface composition

• Composition of interface essentially the same as core

• But % surface area can be quite different!

= different surface/interface areas

Some preferences

prefer

avoid

Ribosome structure• In the nucleolus, ribosomal RNA is

transcribed, processed, and assembled with ribosomal proteins to produce ribosomal subunits

• At least 40 ribosomes must be made every second in a yeast cell with a 90-min generation time (Tollervey et al. 1991). On average, this represents the nuclear import of 3100 ribosomal proteins every second and the export of 80 ribosomal subunits out of the nucleus every second. Thus, a significant fraction of nuclear trafficking is used in the production of ribosomes.

• Ribosomes are made of a small and a large subunit

Large (1) and small (2) subunit fit together (note this figure mislabels angstroms as nanometers)

Ribosome structure• The ribosomal subunits of prokaryotes and eukaryotes are quite similar

but display some important differences.• Prokaryotes have 70S ribosomes, each consisting of a (small) 30S and a

(large) 50S subunit, whereas eukaryotes have 80S ribosomes, each consisting of a (small) 40S and a bound (large) 60S subunit.

• However, the ribosomes found in chloroplasts and mitochondria of eukaryotes are 70S, this being but one of the observations supporting the endosymbiotic theory.

• "S" means Svedberg units, a measure of the rate of sedimentation of a particle in a centrifuge, where the sedimentation rate is associated with the size of the particle. Note that Svedberg units are not additive.

• Each subunit consists of one or two very large RNA molecules (known as ribosomal RNA or rRNA) and multiple smaller protein molecules. Crystallographic work has shown that there are no ribosomal proteins close to the reaction site for polypeptide synthesis. This suggests that the protein components of ribosomes act as a scaffold that may enhance the ability of rRNA to synthesise protein rather than directly participating in catalysis.

• The differences between the prokaryotic and eukaryotic ribosomes are exploited by humans since the 70S ribosomes are vulnerable to some antibiotics that the 80S ribosomes are not. This helps pharmaceutical companies create drugs that can destroy a bacterial infection without harming the animal/human host's cells!

70S structure at 5.5 Å

(Noller et al. Science 2001)

70S structure

30S-50S interface• Overall buried surface area ~8500 Å2

< 37.5 Å2

37.5 Å2 – 75 Å2

> 75 Å2

Protein-nucleic acid Interactions

Interactions in the Ribosome

Calculating interface areas

Given a complex AB:

1. Calculate Solvent Accesible Surface Area (SASA) of A, of B, and of AB

1. SASA lost upon complex formation is

SASA(A)+SASA(B)-SASA(AB)

3. Interface area of A and of B is

(SASA(A)+SASA(B)-SASA(AB))/2

Summary protein(-protein) interactions

• Different binding modes (transient, obligate, also depending on (co)localisation, etc.)

• Hydrophobic patch/hydrophilic rim conferring binding specificity

• Interfaces are physico-chemically positioned in between surface and protein core (amino acid composition, etc.)

• Ribosomes– Small/large subunits, mixture of RNA and protein,

different between prokyarotic and eukaryotic cells (exploited by administering antibiotics), ribosomal protein complexes, protein-RNA binding

today’s lecture

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