Structural Biology: What does 3D tell us?

Stephen J EverseUniversity of Vermont

• Training– PhD & Postdoc with Russell F. Doolittle, UCSD

• structure of fragment D of fibrinogen• structures of double-D of fibrin

– Joined the faculty at UVM in 1998

• Structural biologist (crystallographer)

• Current projects– factor Va– thioredoxin reductase– transferrin

The life of a bio-chemist!!

Everse GroupMaria Cristina Bravo

Brian Eckenroth, Ph.D.

Fundamental Questions

How do protein cofactors modulate enzymes?

What determines and mediates protein-protein

and protein-membrane interactions?

How is a protein’s function defined by

structure?

How does structure prescribe the binding

affinity of a metal?

Coagulation Cascade Factor XIIPrekallikrein

HMW Kininogen“Surface”

Factor XIaHMW Kininogen

MembraneCa2+ Zn2+

Factor IXaFactor VIIIaMembrane

Ca2+

IntrinsicPathway

ExtrinsicPathway

Factor VIIaTissue Factor

MembraneCa2+

Extrinsic Tenase Factor Xa

Factor VaMembrane

Ca2+

Prothrombinase

IX IXa

IX IXa

XI XIa

X XaX Xa

Intrinsic Tenase

ContactActivation Pathway

II IIa“Thrombin”

Ca2+

Ca2+

FXaCa2+

20

FXa

“Prothrombinase”

FVa HC

FVa LC

Ca2+FVa HC

FXa

FVa LC

Ca2+

300,000Prothrombin-Thrombin

ProthrombinaseComponents

Relative Rateof Prothrombin

Activation

FXa 1

FXaCa2+ 2Ca2+

W. Gould @2000

Cu2+

Ca2+

A1

C1C2

A3

Bovine Factor Vai

Funded by:NIHAmerican Society of Hematology

Prothrombinase (Va + Xa)

A3

A1

A2

C2

C1

Hypothetical model

mICA

Eckenroth et al. Protein Science 2010

Outline

• Determining a 3D structure– X-ray crystallography

• Structural elements

• Modeling a 3D structure

Primary Secondary Tertiary Quaternary

Amino acid sequence.

Alpha helices & Beta sheets, Loops.

Arrangementof secondaryelements in 3D space.

Packing of several polypeptide chains.

Given an amino acid sequence, we are interested in its secondary structures, and how they are arranged in higher structures.

Protein Structures

Secondary Structure Helix

• First predicted by Linus Pauling. Modeled on basis x-ray data which provided accurate geometries, bond lengths, and angles. Modeled before Kendrew’s structure;

• 3.6 residues/ turn, 5.4Å/ turn;

• The main chain forms a central cylinder with R-groups projecting out;

• Variable lengths: from 4 to 40+ residues with the average helix length is 10 residues (3 turns).

Secondary Structure The Sheet

• Unlike helix, sheet composed of secondary structure elements distant in structure;

• The strands are located next to each other

• Hydrogen bonds can form between C=O groups of one strand and NH groups of an adjacent strand.

• Two different orientations– all strands run same direction: “parallel”– strands in alternating orientation: the

“antiparallel”.

-Turns• Type I: Also referred to as a turn: H-

bond between Acyl O of AA1 and NH of AA4;

• Type II, glycine must occupy the AA3 position due to steric effects;

• Type III is equivalent to 310 helix;

• Types I & III constitute some 70% of all turns;

• Proline is typically found in the second position, and most turns have Asp, Asn, or Gly at the third position.

Other Secondary Structural Elements

• Random coil • Loop -turn

– defined for 3 residues i, i+1, i+2 if a hydrogen bond exists between residues i and i+2 and the phi and psi angles of residue i+1 fall within 40 degrees of one of the following 2 classes

turn type phi(i+1) psi(i+1)classic 75.0 -64.0inverse -79.0 69.0

• Disordered structure

Viewing Structures

C or CA Ball-and-stick CPK

• It’s often as important to decide what to omit as it is to decide what to include

• What you omit depends on what you want to emphasize

Ribbon and Topology DiagramsRepresentations of Secondary Structures

-helix -strand

N

C

Tools for Viewing Structures

• Jmol– http://jmol.sourceforge.net

• PyMOL– http://pymol.sourceforge.net

• Swiss PDB viewer– http://www.expasy.ch/spdbv

• Mage/KiNG– http://kinemage.biochem.duke.edu/software/mage.php– http://kinemage.biochem.duke.edu/software/king.php

• Rasmol– http://www.umass.edu/microbio/rasmol/

RCSB

http://www.rcsb.org/

GRASPGraphical Representation and Analysis

of Structural Properties

Red = negative surface chargeBlue = positive surface charge

Consurf• The ConSurf server enables

the identification of functionally important regions on the surface of a protein or domain, of known three-dimensional (3D) structure, based on the phylogenetic relations between its close sequence homologues;

• A multiple sequence alignment (MSA) is used to build a phylogenetic tree consistent with the MSA and calculates conservation scores with either an empirical Bayesian or the Maximum Likelihood method.

http://consurf.tau.ac.il/

How do we show 3-D?

• Stereo pairs– Rely on the way the brain processes

left- and right-eye images– If we allow our eyes to go slightly wall-

eyed or crossed, the image appears three-dimensional

• Dynamics: rotation of flat image• Perspective

Stereo pair: Release factor 2/3Klaholz et al, Nature (2004) 427:862

Movies

http://pymol.org

Proteopedia

Protein structures in the

PDBThe last 15 years have witnessed an explosion in the number of known protein structures. How do we make sense of all this information?

blue bars: yearly totalred bars: cumulative total

Classification of Protein Structures

The explosion of protein structures has led to the development of hierarchical systems for comparing and classifying them.

Effective protein classification systems allow us to address several fundamental and important questions:

If two proteins have similar structures, are they related by common ancestry, or did they converge on a common theme from two different starting points?

How likely is that two proteins with similar structures have the same function?

Put another way, if I have experimental knowledge of, or can somehow predict, a protein’s structure, I can fit into known classification systems. How much do I then know about that protein? Do I know what other proteins it is homologous to? Do I know what its function is?

Definition of Domain• “A polypeptide or part of a polypeptide chain that

can independently fold into a stable tertiary structure...”from Introduction to Protein Structure, by Branden & Tooze

• “Compact units within the folding pattern of a single chain that look as if they should have independent stability.”from Introduction to Protein Architecture, by Lesk

• Thus, domains:• can be built from structural motifs;• independently folding elements;• functional units;• separable by proteases.

Two domains of a bifunctional enzyme

Proteins Can Be Made From One or More Domains

• Proteins often have a modular organization• Single polypeptide chain may be divisible into smaller independent

units of tertiary structure called domains• Domains are the fundamental units of structure classification• Different domains in a protein are also often associated with different

functions carried out by the protein, though some functions occur at the interface between domains

1 60 100 300 324 355 363 393

activation domain

sequence-specificDNA binding domain

tetramer-izationdomain

non-specificDNA-bindingdomain

domain organization of P53 tumor suppressor

Rates of Change

• Not all proteins change at the same rate;

•Why?

• Functional pressures– Surface residues are

observed to change most frequently;

– Interior less frequently;

SequenceStructureFunction

Many sequences can give same structure Side chain pattern more important than

sequence When homology is high (>50%), likely to have

same structure and function (Structural Genomics) Cores conserved Surfaces and loops more variable

*3-D shape more conserved than sequence*

*There are a limited number of structural frameworks*

W. Chazin © 2003

Degree of Evolutionary Conservation

Less conservedInformation poor

More conservedInformation rich

DNA seq Protein seq Structure Function

ACAGTTACACCGGCTATGTACTATACTTTG

HDSFKLPVMSKFDWEMFKPCGKFLDSGKLG

S. Lovell © 2002

How is a 3D structure determined ?

1. Experimental methods (Best approach):

• X-rays crystallography - stable fold, good quality crystals.• NMR - stable fold, not suitable for large molecule.

2. In-silico methods (partial solutions -

based on similarity):

• Sequence or profile alignment - uses similar sequences,

limited use of 3D information.• Threading - needs 3D structure, combinatorial complexity. • Ab-initio structure prediction - not always successful.

Experimental Determination of Atomic Resolution Structures

X-ray

X-raysDiffraction

Pattern

Direct detection ofatom positions

Crystals

NMR

RF

RFResonance

H0

Indirect detection ofH-H distances

In solution

• •

Position

Signal

Resolving Power: The ability to see two points that are separated by a given distance as distinct

Resolution of two points separated by a distance d requires radiation with a

wavelength on the order of d or shorter:

d

wavelength

Mark Rould © 2007

Resolving Power

•Lenses require a difference in refractive index between the air and lens material in order to 'bend' and redirect light (or any other form of electromagnetic radiation.)

•The refractive index for x-rays is almost exactly 1.00 for all materials.

∆ There are no lenses for xrays.

nair

nglass

nair

Mark Rould © 2007

X-ray Microscopes?

Scattering = Fourier Transform of

specimenLens applies a second Fourier Transform to the scattered rays to give the image

Mark Rould © 2007

Light Scattering and Lenses are Described by Fourier Transforms

Since X-rays cannot be focused by lenses and refractiveindex of X-rays in all materials is very close to 1.0 how do we get an atomic image?

X-ray Diffractionwith

“The Fourier Duck”

Images by Kevin Cowtanhttp://www.yorvic.york.ac.uk/~cowtan

The molecule The diffraction pattern

Animal Magic

Images by Kevin Cowtanhttp://www.yorvic.york.ac.uk/~cowtan

The CAT (molecule)The diffraction pattern

X-Ray Detector

Computer

Mark Rould © 2007

Solution: Measure Scattered Rays, Use Fourier Transform to Mimic Lens Transforms

A single molecule is a very weak scatterer of X-rays. Most of the X-rays will pass through the molecule without being diffracted. Those rays which are diffracted are too weak to be detected. Solution: Analyzing diffraction from crystals instead of single molecules. A crystal is made of a three-dimensional repeat of ordered molecules (1014) whose signals reinforce each other. The resulting diffracted rays are strong enough to be detected.

A Problem…

Sylvie Doublié © 2000

• 3D repeating lattice;• Unit cell is the smallest unit of the lattice;• Come in all shapes and sizes.

Crystals come from slowly precipitating the biological molecule out of solution under conditions that will not damage or denature it (sometimes).

A Crystal

X-rays

Computer

Crystallographer

Electrondensity map

Model

Scattered rays

Detector

Object

Putting it all together:X-ray diffraction

Sylvie Doublié © 2000

Diffraction pattern is a collection of diffraction spots (reflections)

Rubisco diffraction pattern

3-D view of macromolecules at near atomic resolution.

The result of a successful structural project is a “structure” or model of the macromolecule in the crystal.

You can assign: - secondary structure elements - position and conformation of side chains - position of ligands, inhibitors, metals etc.

A model allows you: - to understand biochemical and genetic data (i.e., structural basis of functional changes in

mutant or modified macromolecule).- generate hypotheses regarding the roles of

particular residues or domains

What information does structure give you?

Sylvie Doublié © 2000

What did I just say????!!!

• A structure is a “MODEL”!!

• What does that mean?– It is someone’s

interpretation of the primary data!!!

So what happens when we can’t get an NMR or X-ray

structure?

2˚ & 3˚ Structure Prediction

Secondary (2o) Structure

Structure Phi (Φ) Psi(Ψ)Antiparallel -sheet -139 +135Parallel -Sheet -119 +113Right-handed -helix +64 +40310 helix -49 -26π helix -57 -70Polyproline I -83 +158Polyproline II -78 +149Polyglycine II -80 +150

Phi & Psi angles for Regular Secondary Structure Conformations

Table 10

- -- -

Secondary Structure Prediction

• One of the first fields to emerge in bioinformatics (~1967)

• Grew from a simple observation that certain amino acids or combinations of amino acids seemed to prefer to be in certain secondary structures

• Subject of hundreds of papers and dozens of books, many methods…

Simplified C-F Algorithm• Select a window of 7 residues

• Calculate average P over this window and assign that value to the central residue

• Repeat the calculation for P and Pc

• Slide the window down one residue and repeat until sequence is complete

• Analyze resulting “plot” and assign secondary structure (H, B, C) for each residue to highest value

Protein Principles

• Proteins reflect millions of years of evolution.

• Most proteins belong to large evolutionary families.

• 3D structure is better conserved than sequence during evolution.

• Similarities between sequences or between structures may reveal information about shared biological functions of a protein family.

The PhD Algorithm

• Search the SWISS-PROT database and select high scoring homologues

• Create a sequence “profile” from the resulting multiple alignment

• Include global sequence info in the profile

• Input the profile into a trained two-layer neural network to predict the structure and to “clean-up” the prediction

http://www.predictprotein.org/

Best of the Best

• PredictProtein-PHD (72%)– http://www.predictprotein.org/

• Jpred (73-75%)– http://www.compbio.dundee.ac.uk/www-jpred/

index.html• SAM-T08 (75%)

– http://compbio.soe.ucsc.edu/SAM_T08/T08-query.html

• PSIpred (77%)– http://bioinf.cs.ucl.ac.uk/psipred/psiform.html

Structure Prediction• Threading• A protein fold recognition technique

that involves incrementally replacing the sequence of a known protein structure with a query sequence of unknown structure.

• Why threading?• Secondary structure is more

conserved than primary structure• Tertiary structure is more conserved

than secondary structureTHREAD

3D Threading ServersGenerate 3D models or coordinates of possible models

based on input sequence

• PredictProtein-PHDacc– http://www.predictprotein.org

• PredAcc– http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/

portal.py?form=PredAcc

• Loopp (version 2) – http://cbsuapps.tc.cornell.edu/loopp.aspx

• Phyre– http://www.sbg.bio.ic.ac.uk/~phyre/

• SwissModel– http://swissmodel.expasy.org/

• All require email addresses since the process may take hours to complete

Ab Initio Folding

• Two Central Problems– Sampling conformational space (10100)– The energy minimum problem

• The Sampling Problem (Solutions)– Lattice models, off-lattice models, simplified chain

methods, parallelism

• The Energy Problem (Solutions)– Threading energies, packing assessment, topology

assessment

Lattice Folding

http://predictioncenter.org/Critical Assessment of protein Structure Prediction (CASP)

http://folding.stanford.edu/

For the gamers out there…

http://fold.it/portal/

Print & Online Resources

Crystallography Made Crystal Clear, by Gale Rhodeshttp://www.usm.maine.edu/~rhodes/CMCC/index.html

http://ruppweb.dyndns.org/Xray/101index.htmlOnline tutorial with interactive applets and quizzes.

http://www.ysbl.york.ac.uk/~cowtan/fourier/fourier.htmlNice pictures demonstrating Fourier transforms

http://ucxray.berkeley.edu/~jamesh/movies/Cool movies demonstrating key points about diffraction, resolution, data quality, and refinement.

http://www-structmed.cimr.cam.ac.uk/course.htmlNotes from a macromolecular crystallography course taught in Cambridge

Evolutionarily Conserved Domains

Often certain structural themes (domains) repeat themselves, but not always in proteins that have similar biological functions.

This phenomenon of repeating structures is consistent with the notion that the proteins are genetically related, and that they arose from one another or from a common ancestor.

In looking at the amino acid sequences, sometimes there are obvious homologies, and you could predict that the 3-D structures would be similar. But sometimes virtually identical 3-D structures have no sequence similarities at all!

The Motif• There are certain favored arrangements of multiple secondary structure elements that recur

again and again in proteins--these are known as motifs or supersecondary structures• A motif is usually smaller than a domain but can encompass an entire domain. Sometimes

the structures of domains are partly named after motifs that they contain, e.g. “greek key beta barrel”

• It should be noted that the term motif, when used in conjunction with proteins, sometimes also refers to sequence features with an associated function, e.g. the “copper binding motif” HXXXXH.

“greek key” motif beta-alpha-beta motif

Limitations of Chou-Fasman• Does not take into account long range

information (>3 residues away)• Does not take into account sequence content

or probable structure class• Assumes simple additive probability (not true

in nature)• Does not include related sequences or

alignments in prediction process• Only about 55% accurate (on good days)

Prediction Performance

45505560657075

CFGOR I

LIMLEVIN

PTIT

JASEP7GOR IIIZHANG

PHD

Scores (%)

An Approach

SAS Calculations

• DSSP - Database of Secondary Structures for Proteins– http://swift.cmbi.ru.nl/gv/start/index.html

• VADAR - Volume Area Dihedral Angle Reporter– http://redpoll.pharmacy.ualberta.ca/vadar/

• GetArea– http://curie.utmb.edu/getarea.html

• Naccess - Atomic Solvent Accessible Area Calculations– http://www.bioinf.msnchester.ac.uk/naccess