The ligand-binding and allosteric pathways of Scapharca HbI James E Knapp Department of Biochemistry and Molecular Pharmacology, The University of Massachusetts Medical School Worcester, Massachusetts

O2O2 O2O2 O2O2 O2O2 MWC Allosteric Models of Cooperative ligand binding O2O2 O2O2 Koshland JMB (1965) 12, Biochemistry (1966) 5, T R T R

1.Show why the R- and T-states differ in ligand affinity. 2.Provide an extremely clear view of the HbI R-state structure. 3.Show how HbI goes from its R-state conformation to its T-state conformation.

Human HbA Scapharca HbII (mollusk) Lamprey HbV (primitive vert) Scapharca HbI (mollusk) Caudina HbD (echinoderm) Urechis Hb (echiurian) Vitreoscilla Hb (bacterium) Rice Hb1 (plant)

Why study Clam HbI? This hemoglobin, because of its simplicity, is an ideal model system to study allosteric processes. p50=7.8 torr, n=1.5

Deoxy HbI – 1.6 Å resolution HbI-CO – 1.4 Å resolution Unligated and CO-ligated Crystal Structures

Ligand Affinity in Heme Proteins 1.Modulate the reactivity of the heme iron. 2.Control access of the ligand to its binding site. 3.Stabilize the bound ligand.

Ascaris Hemoglobin Myoglobin Proximal Contributions to Ligand Affinity PNAS (1995) 92, Nature (2000) 403, KO 2 =220  M -1 KO 2 =1.1  M -1

PNAS (1995) 92, Structure Fold Des. (2000) 8, E7 Rice Hb (ligand access) Ascaris Hb (O 2 stabilization) B10 E7 Distal Contributions to Ligand Affinity

Cooperative proteins use one or more of these three types of mechanisms to either increase the oxygen affinity of the R-state and/or decrease the ligand affinity of the T-state.

1. The conformation of Phe 97 directly alters ligand affinity via proximal effects. 2. The position of the heme group modulates ligand affinity through distal effects. 3.The movements of the heme group and Phe 97 are tightly coupled together. 4.The quaternary structural change and heme group movements are coupled together. Mutagenesis Results: Why are the R- and T-states different?

p50 O 2 (torr) n O 2 off rates O 2 on rates WT R < TR > T F97L1.01.2R=TR > T F97A F97Y R=TR > T The F97A, F97L, and F97Y mutations abolishes the proximal contributions to cooperativity.

F97Y p50=0.08 torr n=1.1 Off rates R=T On rates R >T

1. The conformation of Phe 97 directly alters ligand affinity via proximal effects. 2. The position of the heme group modulates ligand affinity through distal effects. 3.The movements of the heme group and Phe 97 are tightly coupled together. 4.The quaternary and heme group movements are coupled together. Mutagenesis Results: Why are the R- and T-states different?

p50 (torr)n WT I114F Mutation of Ile 114 to Phe attenuates ligand-linked heme movement, lowering oxygen affinity and nearly abolishes cooperativity Phe 114 Ile 114 Phe 97

2.1 Å The distal histidine sterically restricts ligand binding when the heme group assumes its T-state position. Superposition of CO-HbI (grey) onto deoxyHbI (black)

Å resolution Rfactor=0.201 Rfree= Å resolution Rfactor=0.166 Rfree=0.210 Unligated and CO-ligated H69Q H69Q: p50=75 torr, n=1.3 H69Q, F97L: p50=4 torr, n=0.95

1. The conformation of Phe 97 directly alters ligand affinity via proximal effects. 2. The position of the heme group modulates ligand affinity through distal effects. 3.The movements of the heme group and Phe 97 are tightly coupled together. 4.The quaternary structural change and heme group movements are coupled together. Mutagenesis Results: Why are the R- and T-states different?

Ultra-high resolution Crystallography 1.The stereochemistry of ligand binding reflects ligand affinity. 2.Show that the R-state is a set of sub-states and alternate conformations 3.Demonstrate that the anisotropic motion of the heme group and Met 37 are consistent with our current mechanism for the allosteric transition that occurs in HbI. 4. Observe Hydrogen atoms within HbI.

Unligated HbIHbI-O 2 HbI-CO Resolution Å Å Å Reflections Observations Completeness (%) Rsym (%) Resolution Å40.0 – 0.78 Å Å Reflections Observations Completeness (%) Rsym (%) Ultra-high Diffraction Data Statistics

HbI-CO Å R=16.4% HbI-CO HbI-O Å R Free = 13.5% R=11.9% Fe-O1-O2: 126.2°, 128.8° Fe-C-O: 173.3°, 173.5° Fe-O1: 1.82 Å, 1.84 Å Fe-C: 1.74 Å, 1.77 Å Fe-N  2: 2.10 Å, 2.11Å Fe-N  2: 2.06 Å, 2.07 Å Å R Free = 14.2% R=12.4%

Major Conformation Minor Conformation Pre A helix, B subunit EF turn, A subunit 1 Pro 5 Asp 9 Gln 4 Tyr 2 Ser 3 Val 6 Ala 7 Ala 8 Ala 87 Pro 84 Leu 88 Asp 86 Asn 85 Asp 83 Gln The R-state includes a population of sub-states, even in the crystal lattice.

Sub-states are present at the subunit interface, including alternate water positions. Phe A97 Phe B97 2Fo-Fc map contoured at 2.0  2Fo-Fc map contoured at 1.0 

Protein motion in HbI relates to ligand- linked structural changes. 2Fo-Fc map, contoured at 1.2  Met 37 Anisotropic motion modeled as thermal ellipsoid spheres

Time Resolved Crystallography T R T R R-state HbI T-state HbI 2CO

40.0 – 1.4 Å resolution, 1  Contour 40.0 – 2.5 Å resolution, 1  Contour 40.0 – 2.8 Å resolution, 0.9  Contour 97F 101H 69H 96’K 37M 97F 69H 101H 97F 96’K Biochemistry (2003) 42, Full and reversible ligand-induced allosteric structural transitions can occur in HbI-CO crystals

CCD Dye laser Heat load shutter 2  s chopper ms shutter Experimental Setup for Time-resolved Crystallography e-e- BIOCARS, APS

Crystals and Data Collection Sample Diffraction for HbI Dithionite + Phosphate Polyvinyl film over HbI crystal Cement

Ligand Migration in HbI 37M 69H 121V 122L 135W 36L L73 114I A B BEG

1ns H101 H69 M37 W135 I25 BEG B Ligand migration in HbI: dissociated CO moves to the B site within 1ns (Fo light -Fo dark : Red: -3 , Blue: 3  )

Ligand migration in HbI: dissociated CO moves from distal pocket to BEG channel within 10ns (Fo light -Fo dark : Red: -3 , Blue: 3  ) 25ns H101 H69 M37 W135 I25 BEG

Mutation of Met 37 to Val (M37V) lowers geminate rebinding as the ligand migrates to a B-site in the distal pocket V37 H69 H101 F97 (Red: -3 , Blue: 3  ) B-site M37V Ligand binding Kinetics ns

The allosteric transition of HbI goes through a transient intermediate that is distinct from both the R and T-states. The allosteric transition involves the movement of the heme group before the conformational change of Phe 97.

5ns25ns50ns100ns300ns800ns 3s3s green: 5  blue: 2.5  red: -2.5  M37V Heme A movement

Phe 51 Phe 97 His 101 M37V-CO – gray 50ns intermediate – cyan deoxy HbI - magenta Early transitions of distal residue Phe 51 may contribute to enlargement of proximal cavity for T-state packing of Phe 97

5ns25ns50ns100ns300ns800ns 3s3s Phe 41 Phe 51 Heme buckling results in a transient displacement of distal residue Phe 51 M37V-CO – gray 50ns intermediate – cyan deoxy HbI - magenta blue: 3 

5ns25ns50ns100ns300ns800ns 3s3s Structural transitions on the Proximal side of the Heme His 101 Phe 97 Asn 100 M37V-CO – gray 50ns intermediate – cyan deoxy HbI - magenta dark blue: 3  light blue: 2  red: -3 

, Kinetics of the Allosteric Transition within HbI

5ns25ns50ns100ns300ns800ns 3s3s Bulk Solvent Subunit Interface dark blue: 3  red: -3  Gate to water channel

The Kinetics of the hydration of the T-like interface as measured by the disappearance of the water molecules that border the interface Log (ns)  Water Density/  photolysis Density

Our model showing cooperative ligand binding in HbI 2O 2 TR I

Summary 1.Ligand-linked movement of the heme group causes a distal effect on oxygen affinity whereas the ligand-linked Phe 97 movement induces a proximal effect. These two conformational changes are coupled together. 2.The sterochemistry of a ligand-heme complex is consistent with the reactivity of the heme group for that ligand. 3.Allosteric transitions proceed through at least one kinetic intermediate, characterized by an unligated, buckled heme packed tightly in an R-state subunit. 4.Movement of Phe 97 to its T-state position lags heme movement considerably. The change in the water structure lags the movement of Phe 97.

University of Massachusetts, Worcester William Royer Kristen Strand Hitesh Sharma Betha Komali Animesh Pardanani Michele Bonham Candace Summerford Lauren Cushing Rice University & UMass Quentin Gibson BIOCARS Keith Moffat Vukica Srajer Reinhard Paul ESRF Michael Wulff NIH, AHA

University of Massachusetts, Worcester William E. Royer Jr. Quentin Gibson Betha Komali Univ. of Chicago – BioCARS Vukica Šrajer Reinhard Pahl NIH, AHA

Laue Diffraction Statistics WTM37V 1ns5ns25ns 300ns Completness (%) dark to 2.2 Å light to 2.2 Å wRmergeF 2 (%) dark data set light data set Photolysis Aheme (%) Photolysis Bheme (%) Min Difference peak      Max Difference peak 8.5  6.7  8.5  8.1 

5ns25ns50ns100ns300ns800ns 3s3s green: 5  blue: 2.5  red: -2.5  M37V Heme B movement

HbI WT G=45% NO=20 L73V G=55% NO=22 L73F G=23% NO=10 I114M G=27% NO=10 L73M G=23% NO=15 I114F G=60% NO=2 Structural and kinetic analysis of HbI with mutations at postions 73 and 114. G= geminate O 2 rebinding, NO=nitric oxide on rate

Dithionite + Phosphate Polyvinyl film over HbI crystal Cement Before After Mounting HbI Crystals for Laue Experiments Cement

Timing scheme for ns time-resolved experiments at BioCARS 14-ID beamline with hybrid mode of the APS storage ring.

F41L is thought to lower ligand affinity by lowering the energy barrier to reach the R-state like intermediate structure. M37F ocupies two conformations, one of which increases the energy barrier for HbI to reach its R-state like intermediate structure. F41W, F51L, and F51W all test the same hypothesis. Mutations that test the intermediate structure

A = B Cooperativity or Homotropic interactions A ≠ B Heterotropic interactions

1. Add sample to a tonometer with the ligand of interest. 2. Flash sample to disrupt the iron-ligand bond. 3. Monitor A 418 to determine the rate of ligand rebinding in the Hb sample. Ligand-Binding Kinetics

   

Ligands Move from the ligand binding or A-site to the B-site (Black) and then to BEG site (red).

Despite structural changes limited to the interface water structure, mutation of Thr 72 to Val results in a 50-fold increase in oxygen affinity: p50 (torr)n WT T72V0.21.7

Raising the osmotic pressure increases the oxygen affinity of HbI

Human Hemoglobin - HbA    

Ligands move to the B-site upon photolysis and then to a cavity defined by the B, E, and G helices. The M37V substitution alters the kinetics of ligand migration, but not the pathway.

Initial HbI movements following ligand release R-stateTS #      I1I1 Met 37 Ile 114 His 69 His 101

Ligation of HbI results in extrusion of Phe 97 from the proximal pocket Distal His Distal His ligand Proximal His Phe 97 Proximal His

Laue Diffraction Statistics WT M37V 1ns5ns25ns 300ns Completness (%) dark to 2.2 Å light to 2.2 Å wRmergeF 2 (%) dark data set light data set Photolysis Aheme (%) Photolysis Bheme (%) Min Difference peak      Max Difference peak 8.5  6.7  8.5  8.1 

CCD laser Heat load shutter 2  s chopper ms shutter

Mutation of Phe 97 in Scapharca HbI leads to increased oxygen affinity and sharply diminished cooperativity in equilibrium oxygen binding experiments: p50 (torr)n Wild-type F97L F97A F97Y

Testing the Swinging His Model p50 (torr)nkineticsCD H69A--no H69V---- H69L--no- H69F--nocrystals H69S--no H69N--no H69Q751.3some(structure)

Ile 114 Phe 114 Phe 97 Mutation of Ile 114 to Phe attenuates ligand-linked heme movement, lowering oxygen affinity (p50=21 Torr) and nearly abolishes cooperativity (n=1.1).

Proximal Contributions to cooperativity involve Phe 97 and His Hydrogen bond between Phe 97 O and His 101 N  1 increases the ligand affinity of the R-state. 2.This hydrogen bond is weakened in the T-state, thereby lowering the ligand affinity. 3.The T-state conformation of the Phe 97 arromatic ring will impede the movement of the heme iron, thereby lowering the ligand affinity of the T-state. This barrier is not present in the R-state.

N C Ligands exit Mb when the distal histidine swings out, opening a direct route to the solvent. Ligand Migration in Sperm Whale Myoglobin K30D ka=10 3, G=35%, no=35  m s -1

Mutations that Block Ligand Entry and Exit 1.I25W cloned, expressed, crystallized 2.I25M needs to be made 3.L36W in progress 4.I118W needs to be made 5.I118M needs to be made