structural biology: what does 3d tell us? stephen j everse university of vermont
TRANSCRIPT
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