CURRENT TOPICS IN

STRUCTURAL BIOLOGY

BS409/7109

“Current Topics in Structure as a Tool for Biologists” Learning Objective The course objective is to acquire a firm grounding in structural biology that can serve as a tool for both understanding the basis of biological activity as well as conducting research in the biological sciences. Content Biology students of today are commonly faced with challenges that require an understanding of physics and chemistry, because contemporary research in the biological sciences is highly interdisciplinary and is becoming only increasingly more so. To answer the most important biological questions of today, one must generally entertain multiple methodologies with the strongest coverage coming from addressing, for example, structural, molecular and cellular angles of a problem at the same time. As such, this course is aimed at providing an in depth understanding of three-dimensional macromolecular structure and the relationship between the conformation of proteins and nucleic acids with the biological activities of recognition, transport, signalling, and catalysis. We cover timely issues related to multi-molecular assemblies, catalytic machines, and membrane proteins, utilizing computer graphics for a profound visualization of biological function. Learning Outcome 1) Students can visualize and analyze macromolecular structures using molecular graphics

software. 2) Given the known function of a macromolecule (protein, DNA, RNA or protein-nucleic

acid complex), the student can conduct an assessment of the molecule in 3-D to understand the structural and chemical basis of its activity.

Student Assessment Students will be assessed by: a. Final 2.5-hour written examination (70%) b. Computer graphics exercises completed in a lab notebook (30%)

References 1) Protein Structure and Function; Petsko & Ringe; 2004; New Science Press. 2) Principles of Nucleic Acid Structure; Neidle; 2008; Academic Press. 3) Biochemistry; Voet, Voet; 2011; John Wiley & Sons. 4) Bioinorganic Chemistry (A Short Course); Roat-Malone; 2007; John Wiley & Sons. 5) Introduction to Macromolecular Crystallography; McPherson; 2009; John Wiley & Sons.

Lecture Schedule

BS409 - Dr Curt Davey Date (1430-1720)Lecture Title Lecturer

Week 1 Macromolecular Structure and Analysis Curt Davey Aug. 13Week 2 Methods in Structural Biology and Some Recent Applications Julien Lescar Aug. 20Week 3 The Use of X-ray Crystallography for Antiviral Drug Discovery Julien Lescar Aug. 27Week 4 Ion Channels & Molecular Discrimination Konstantin Pervushin Sep. 3Week 5 Membrane-Associated & Other Hot Enzymes (‘Memzymes’) Konstantin Pervushin Sep. 10Week 6 Molecular Interactions by NMR spectroscopy Surajit Bhattacharyya Sep. 17Week 7 Nucleic Acid Form and Activity Curt Davey Sep. 24

Week 8 Structural Enzymology Zhao-Xun Liang Oct. 8Week 9Week 10 Electron Microscopic Analysis of Large Macromolecular Assemblies Sara Sandin Oct. 22Week 11 Structural Biology of Stress Pathways in Cancer Pär Nordlund Oct. 29Week 12 DNA Recognition & Manipulation Curt Davey Nov. 5Week 13 Nucleosome Formation & Function Curt Davey Nov. 12

Term Break

A Brief Early History of Structural Biology:

1953: Determination of general structure of DNA

1958: First structure of a protein (myoglobin)

1965: First structure of an enzyme (lysozyme)

1974: First structure of an RNA biomolecule (tRNA)

1979: First (atomic) structure of a DNA molecule (Z-DNA !)

1984: First (atomic) structure of a membrane protein (photosyn. rxn. center)

1986: First structure of a protein-DNA complex (EcoRI + DNA)

Techniques for Structural Characterization of Macromolecules: Optical spectroscopic methods (CD, Raman, ESR…) details on specific molecular attributes Cryoelectron micoscopy resolution limit ~10 Å Electron crystallography resolution limit ~4 Å Neutron crystallography H locations; but requires very large crystals NMR (Nuclear Magnetic Resonance) H locations + dynamics; ~20 kD limit, low data:parameter # X-ray crystallography typical resolution- 2 to 3 Å; very large systems also

CTSB – Lecture I C.A. Davey

Macromolecular Structure

& Analysis

Philosophy In order to understand structure, you need to know your chemistry. Chemistry is just chemistry… but structure is structure plus chemistry. If you know your chemistry, you will have good chemistry with structure… and the experts say everyone needs structure in their lives, so here we are.

How do we maximize our ability to get the most out of structures ?

Bring in your chemistry (bonding, reactivity) and physics (thermodynamics, kinetics)

so that you really have ‘vision’ when looking at 3-D structures…

Chemical interactions that govern macromolecular structure/stability

(van der Waals attraction)

Ice

Water structure & the wonderful H-bond

In the strongest, ideal, H-bonds the donor-hydrogen and acceptor-lone pair groups are linear. This means the hydrogen-donor ‘+’ dipole is pointing directly at the electrons of a lone pair.

Acceptor (lone pair)

Hydrogen

Donor

Even with super high resolution X-ray data (1.55 Å below) can NOT see Hydrogen atoms

Model built from electron density H atom positions inferred from C/N/O

Not knowing H positions: Planar H-bonding groups (*)

*

* * *

* * *

H atom locations are known from the C/N/O positions for planar groups

major

major

minor

minor

As for proteins, H atom positions generally not known from X-ray data.

However, bases are almost completely planar, thus H atom positions are more or less known from

non-H atom structure. Importantly, all H-

bonding with bases is essentially planar.

Not knowing H positions: Ambiguity in donor vs. acceptor assignment (*)

*

* * *

*

Ser

Ser

Thr

Not knowing H positions: General ambiguity in protonation state at physiological pH(*)

*

???

uncharged !

At typical resolutions for protein structures, ~2 to 3 Å: Orientations of Asn, Gln, and His side chains ambiguous from electron density alone…

GLN

2.92 Å

3.25 Å

3.50 Å ?

Solvent liberation drives folding and macromolecular interactions

Water molecules have a constrained ordering– low entropy– around phobic surfaces (look left). Generally, burying any surface residues, but especially phobic ones, will liberate ordered solvent. The trick for macromolecular folding and recognition is to optimize interactions in the folded or complexed states.

ASA = solvent Accessible Surface Area Change in total surface area: ∆ASA = [ ASA1 + ASA2 ] - ASAcomplex

The general underlying driving force behind macromolecular folding and complexation comes from desolvation of the interacting surfaces. ‘Liberation’ of bound water molecules into the bulk solvent gives rise to a favorable free energy contribution by increasing the solvent entropy. This effect is greatest for hydrophobic (‘phobic’) surfaces that become buried upon complex formation.

-∆Ssolvent is usually favorable (-) &

-∆Smacromol is usually unfavorable (+) :

∆Gcomplex = ∆Gmacromol + ∆Gsolvent

“ = ∆Hmm - T∆Smm + ∆Hsol - T∆Ssol

∆ASA = 3400 Å2

Desolvation (∆ASA) can give rise to a general driving force for complex formation, but well-defined (‘optimally fitted’) polar and VDW contacts generate specificity as well as stability.

The phobic effect is the generally driving force for protein and nucleic acid folding. Most proteins have buried phobic clusters (left). However, the optimal packing of phobic side chains minimizes volume and thus void regions (the “pressure X volume” term in enthalpy). Thus, tight packing is critical, and even boring phobic side chains have electrostatics.

Phe

Phe Phe

Face-to-face overlap bad. Edge-to-face good.

π-H

The π-Cation

Interaction

More hidden

charges...

Up to almost +/- 1.0 unit of charge at a helix terminus involved in strong electrostatic interaction !

The Helix

Dipole

ASP

GLN

Histone-DNA interactions in the nucleosome: Helix dipole gives charge-assisted hydrogen bonds between the peptide NH groups at the N-termini and phosphate groups (circled). This promotes very strong and accurate positioning in DNA-histone binding.

Chloride anion-hydrate binding in the nucleosome: Anions (magenta) can associate with ‘free’ peptide NH groups at the helix N-termini. Free meaning no side chain capping or binding interactions.

C-terminus

Divalent metal-hydrate binding in the nucleosome: Conversely, cations (magenta) are attracted to ‘free’ peptide C=O groups at the helix C-termini. Free still meaning no side chain capping or binding interactions.

Potassium (left) and chloride (below) channels exploit helix dipoles too…

Analogous to the K+ channels (bottom), the Cl- channels (top) facilitate ion passage by directing Cl- ions into the channel through focusing the positive ends of the dipoles of 2 α-helices towards the center of the channel.

H-bonding preferences

H-bonding

Van der Waals

Van der Waals

* Bifurcated H-bonding (‘sharing’)

* *

METAL CATIONS

* present in ~half of all proteins

* required for function of DNA/RNA

* many errors in PDB

ubiquitous

trace (many others)

Site-Selectivity of Cation-Macromolecule Binding

1) Coordination Geometry Ligand geometry selects for suitable cation type ** M+: more variable compared to M2+

** M2+: well-defined / constant ligand number 2) Free Energy of Dehydration ∆Gdehyd proportional to: {ion charge}2 & {ion radius}-1

** most difficult to dehydrate small/highly charged ions Mg2+ > Mn2+ > Ca2+ >>> Na+ > K+ > Cs+ 3) Electrostatic Coordination Energy Does ligand charge density compensate for ion dehydration ? ** charge-based discrimination ( M+ vs. M+2 ) ** ionic radius-based discrimination ( Na+ vs. K+ ) 4) Softness of Ion & Ligand Ligands select for ions of similar softness ** Softness Polarizability Covalency of Ion-Ligand Bond Hard Cations: Na+, K+, Mg+2 > Ca+2 > Mn+2 Soft Cations: Cu+, Cd+2, Pt+2, Hg+2

Selectivity between

ubiquitous metals

Selectivity between trace and

ubiquitous metals

Na Channel 5 or 6 ligands: Sometimes carboxylate (-1) group, selects for higher charge density of Na & compensates for greater ∆Ghyd of Na

K Channel 8 ligands: Always uncharged groups, select for lower charge density of K

*** This just means that hard species interact purely electrostatically, while soft species form (partial) covalent bonds through orbital mixing/interaction and charge transfer. (This is supplementary and just for your information.) ***

Increasing polarizability

‘Hard’

‘Soft’

hard hard

hard

hard hard

hard

hard

soft

soft

soft

Zn binding sites, often serving a purely structural role, utilize a variety of different protein configurations. However, all have soft ligands with tetrahedral coordination geometry.

Example PDB structure files, for an intricate enzyme and a protein-enzyme complex with a large interface:

Human myeloperoxidase 1CXP

CDK2-Cyclin 1FIN

http://www.rcsb.org/pdb

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