Conformation of Ligands on Macromolecules

• Introduction: Diversity of Complexes Studied by NMR

• Influence of Chemical Exchange on NMR Spectra of Receptor-Ligand Complexes

– Fast vs. Slow Exchange

• Characterizing Exchange Rates by NMR

• NMR Techniques for Studying Receptor-Ligand Interactions and Ligand Conformations on Receptors

– Deuteration– Isotope Filtering– Transfer NOE

References

General

Craik, D. J. and J. A. Wilce (1997). “Studies of protein-ligand interactions by NMR.” Methods in Molecular Biology 60: 195-232.

Methods of Enzymology 239 (1994)Section IV, pp. 657-767, Protein-Ligand Interactions

Feeney, J. and B. Birdsall (1993). NMR Studies of Protein-Ligand Interactions. NMR of Macromolecules: A Practical Approach. G. C. Roberts, Oxford: 183-215.

Wand, A. J. and S. W. Englander (1996). “Protein complexes studied by NMR spectroscopy.” Current Opinion in Biotechnology 7(4): 403-8.

Ligand Binding and Chemical Exchange

“Biomolecular NMR Spectroscopy” by Evans (1995)Chapter 1.3.1 – 1.3.3, pp. 43-46, Kinetics

Lian, L. Y. and G. C. Roberts (1993). Effects of Chemical Exchange on NMR Spectra. NMR of Macromolecules: A Practical Approach. G. C. Roberts, Oxford: 153-182.

Isotope Filters

Breeze, A. L. (2000). “Isotope-filtered NMR methods for the study of biomolecular structure and interactions.” Progress in Nuclear Magnetic Resonance Spectroscopy 36(4): 323-372.

Gronenborn, A. M. and G. M. Clore (1995). “Structures of protein complexes by multidimensional heteronuclear magnetic resonance spectroscopy.” Critical Reviews in Biochemistry & Molecular Biology 30(5): 351-85.

Otting, G. and K. Wuthrich (1990). “Heteronuclear filters in two-dimensional NMR” Quarterly Reviews of Biophysics 23(1): 39-96.

Transferred NOE

“Biomolecular NMR Spectroscopy” by Evans (1995)Chapter 6.5, pp. 246-247, Transferred NOE

Ni, F. (1994). “Recent Developments in transferred NOE methods.” Progress in Nuclear Magnetic Resonance Spectroscopy 26: 517-606.

Campbell, A. P. and B. D. Sykes (1993). “The two-dimensional transferred nuclear Overhauser effect: theory and practice.” Annual Review of Biophysics & Biomolecular Structure 22: 99-122.

Deuteration

LeMaster, D. M. (1990). “Deuterium labelling in NMR structural analysis of larger proteins.” Quarterly Reviews of Biophysics 23(2): 133-74.

Diversity of Complexes Studied Using NMR

• protein-peptideCD4 36-59 peptide/HIV gp120 (transfer noe)mellitin peptide/calmodulin (deuteration)pY peptide from PDGF receptor/SH2 of PLCC (isotope filter)

• receptor-ligandcyclophilin/cyclosporin A (isotope filter)FKBP/FK506 (isotope filter)

• enzyme-substrate/inhibitorNAD+/lactate dehydrogenase (transfer noe)glutathione/glutathione transferase (transfer noe)

• protein-carbohydrateSlex tetrasaccharide/E-, P-, L-selectin (transfer noe)

• antibody-antigenFab/Fv fragment

• protein-nucleic acidhomeodomain/DNA fragment (isotope filter)U1A protein/ RNA hairpin (isotope filter)

• protein-proteinp53 tetrameric oligomerization domain (isotope filter)

E + L ELkon

koff

where: kon is a bimolecular rate constant that is a measure of probability of productive encounter between free receptor and ligand – kon is limited by diffusion controlled rate 107 – 108 M-1 s-1

koff is a unimolecular rate constant that is inversely proportional to – the lifetime of the complex

The binding affinity is given by equilibrium dissociation constant:

KD = [E][L]/[EL] = koff/kon

As a rough approximation:• tight binding (slow exchange) is characterized by KD << M• weak binding (fast exchange) is characterized by KD >> M

Binding Equilibria

Effect of Exchange on NMR Spectra

Suppose a nucleus is able to experience two different environments, what would its nmr signal look like?

H1.5ppm 1.0ppm

1.5ppm 1.0ppm

If exchange between the two environments was turned off, we would see two signals – the time domain signal or FID would be a superposition of the FID of each signal

The frequency domain spectrum is a superposition of the individual spectra.

low freq. component = 1.5ppm signal

high freq. component = 1.0ppm signalFT

H

Slow Exchange

What happens if we turn exchange between the two sites on?

1.5ppm 1.0ppm

1.5ppm 1.0ppm

If exchange between the two environments is very slow (lifetime of complex is long):

kex << the frequency (or chemical shift) of each environment is sampled before exchange

The frequency domain spectrum contains two signals representing a superposition of the individual signals (the intensities of the signals would represent the relative amounts of each species).

low freq. component = 1.5ppm signal

high freq. component = 1.0ppm signalFT

exchange event

HH

Fast Exchange

1.5ppm 1.0ppm

1.25ppm

If exchange between the two environments is very fast (lifetime of complex is short):

kex >> each sampled point is a weighted average of the points from the free and bound states

The frequency domain spectrum contains a single signal whose chemical shift is a weighted average of the chemical shifts of the free and bound states:obs = pfree*free + pcomplex*complex where p represents mole fraction

FT

H

weighted average of free and bound points

H

Intermediate Exchange

As the lifetime of complex approaches the frequency difference of the signals corresponding to the free and bound states:

kex ~

the signals become “exchange broadened”:

slow exchange limit

fast exchange limit

intermediate exchange

Before we attempt to study the structure of a ligand in a complex, it is useful to determine if the complex is in fast or slow exchange on the NMR timescale. This is often done by titrating the ligand (L) into a sample containing the receptor (R) and monitoring the NMR spectrum:

Titration under conditions of:slow exchange fast exchange

[L]:[R]

0:1

0.5:1

1:1

1.5:1

2:1

Rf Rb Lf Lb Rf Rb Lf Lb

Characterizing Fast/Slow Exchange by NMR

NMR Techniques for Determining the Structure of a Ligand in a Complex

To determine the structure of a bound ligand using NMR methods, we must be able to assign the signals belonging to the ligand within the complex and determine structural constraints belonging to those signals (ie. NOEs). To determine the structure of a ligand bound to a receptor, we must be able to distinguish the ligand signals from receptor signals:

How to separate signals of receptor from those of ligand within the complex?? To distinguish ligand signals from receptor signals, the following techniques are available:

o DEUTERATION

o ISOTOPE LABEL/FILTER

o TRANSFER NOE

+ =

spectrum of ligand spectrum of receptor spectrum of complexNote: not necessarily a superposition of ligand and receptor spectra

Deuteration of Receptor

replacement of the protons by deuterons in the receptor will eliminate the signals of the receptor within the complex:

Deuteration of the receptor would normally be used to study the structure of a ligand in a complex under conditions of slow exchange. This method has largely been superseded by isotope filtering methods because:

expense of deuteration ability to edit spectra of complexes for the ligand signals or receptor signals in isotope filtering experiments exchangeable protons on receptor (ie. amide protons) are not removed from spectra collected in water

spectrum of ligand bound to deuteratedreceptor

deuteration of receptor

spectrum of complex

1H1H 1H2H

Hsu, V. L. and I. M. Armitage“Solution structure of cyclosporin A and a nonimmunosuppressive

analog bound to fully deuterated cyclophilin.” Biochemistry 31, 12778 (1992).

objective: determine the structure of the immunosuppressive drug cyclosporin bound to the immunophilin cyclophilin.

complex: Cyclosporin A (CsA, cyclic 11-mer peptide) + cyclophilin (CyP, 17.7 kDa)

background: • cyclosprin A exhibits immunosuppressive activity (prevents organ/bone marrow transplant rejection). • CyP, a cytosolic protein immunophilin, binds to CsA, and has proline cis-trans isomerase activity that is inhibited by CsA.• Structures reported for CsA in crystal and in CHCl3 solution indicate presence of a cis peptide bond. Is cis bond an important feature of complexed CsA?

complex: Cyclosporin A (CsA, cyclic 11-mer peptide) + cyclophilin (CyP, 17.7 kDa)

binding: KD ~ 10-8 M

sample and methods: • perdeuterated CyP by recombinant expression in bacteria grown in deuterated algal hydrolysate in D2O• NMR sample consisted of 0.4mM complex with excess CyP• 2D homonuclear experiments run (COSY,TOCSY,NOESY)• 66 intraresidue and 55 interresidue NOEs obtained and used in XPLOR (simulated annealing protocol)

result: • structures generated had 0.54A rmsd for backbone• bound structure contains only trans peptide bonds and shows no regular secondary structure (free CsA in organic solvents and in the crystal has a sheet structure)• intermolecular nOes were identified by comparing NOESY spectra of the CsA/[2H]-CyP complex with a fully protonated complex – supports prediction that the CyP Trp indole ring is located in the binding site.

spectrum of complex

Isotope Filtering

labeling of the receptor (or ligand) with 13C/15N permits the use of an “isotope filtering” nmr experiment to select for signals in the spectrum from protons that are either bonded to 13C/15N or 12C/14N:

spectrum of ligand

isotope filter to select for 12C/14N

1H1H

13C/15N 12C/14N

1H

12C/14N

1H1H

13C/15N 12C/14N

spectrum of complex

isotope filter to select for 13C/15N

spectrum of receptor

1H

13C/15N

The Isotope Filter

phase x x-x

x x x-x or x x

selects X-1H

selects for 1H that are not

X-1H

1H

X

1/2JXH receiver1/2JXH

RARBLA LB

RA

RB

LA

LB

If the receptor (R) or ligand (L) is isotopically labeled, an isotope filter at each end of a NOESY can be use to select for:

NOEs between protons on the ligand ( ) NOEs between protons on the receptor ( ) NOEs between ligand protons and receptor protons ( )

depending on the phase cycling scheme used. Isotope filtering can be used to study the structure of a ligand in a complex under conditions of fast or slow exchange.

2D NOESY spectrum

In an isotope filtered experiment, a 90o pulse is replaced by:

R. R. Rustandi, D.M. Baldisseri, and D. J. Weber “Structure of the negative regulatory domain of p53 bound to

S100B()” Nature Structural Biology 7, 570 (2000).

objective: determine the structure of p53 peptide – S100B() complex to gain insight on how to develop inhibitors that block the Ca2+ dependent interaction between the two proteins.

complex: peptide derived from p53 (22 residues, S367-E388) bound to S100B() (~13 kDa) forms a quaternary complex consisting of two p53 peptides per S100B() dimer.

background: • the tumor suppressor protein p53 is a transcription activator that signals for cell cycle arrest and apoptosis• the c-terminal negative regulatory domain (residues 367-392) contains a site that activates p53 when phosphorylated• members of the S100 protein family are overexpressed in tumor cells and their function is tightly regulated by Ca2+ concentrations• upon binding Ca2+, S100B() undergoes a large conformational change which exposes a hydrophobic patch that is required for binding to p53• the Ca2+ dependent interaction of p53 with dimeric S100B() causes inhibition of p53 dependent transcription

binding: KD ~

sample and methods:• 3-6 mM [13C/15N]-S100B() + 5-10mM unlabeled peptide with 6-13mM Ca2+

• asymmetrically labeled S100B(): 50% unlabeled S100B() + 50% [13C/15N]-S100B() • peptide + [2H]-S100B()• assign peptide in complex using 15N-filtered & 13C-filtered NOESY/TOCSY and 1H-1H NOESY/TOCSY on peptide + [2H]-S100B()

result: • determine and compare S100B() structures in the apo, Ca2+ and p53 peptide bound states• p53 peptide is random coil in absence of S100B() but adopts a helical conformation when bound to Ca2+ loaded S100B()

The Transfer NOE

The transfer NOE is typically used to study the structure of a ligand in a complex under conditions of fast exchange. No isotopic labeling of the ligand or receptor is necessary. Even under conditions where there is only a small percentage of bound ligand in solution, the “memory” of NOEs present within the bound state conformation of the ligand are carried over to the ligand free in solution under conditions of fast exchange:

HaHa

Hb

Hb

2D NOESY spectrumbefore addition of receptor

HbHa

Hb

Ha

HbHa

Hb

Ha

2D NOESY spectrumafter addition of small amount

of receptor (a few percent)

objective: Determine the gp120-bound conformation of CD4 to better understand the gp120-CD4 receptor interaction at the molecular level and help elucidate the mechanism of HIV entry into the cell.

complex: CD4 36-59 peptide with HIV-1 gp120. The peptide is known to block the interaction with gp120.

background: • The HIV-1 gp120 is a viral surface glycoprotein that binds to CD4 receptor and allows the virus to gain entry into T-lymphocyte CD4+ cells.• Peptide of human CD4 receptor (435 residues) - structures of two different crystal forms are different. • Peptide is random coil free in solution.

binding: KD ~ 10-9 of extracellular domain of receptorKD ~ 100-500 M for peptide

methods:• NMR sample consisted of 2mM peptide and 66M gp120• NOESY at 6 different mixing times• simulated annealing with 107 constraints (4 long-range and 42 medium range)

Gizachew et al. “NMR studies on the conformation of the CD4 36-59 peptide

bound to HIV-1 gp120”Biochemistry 37, 10616-25 (1998)

result: • structures different than those found in crystal• see decreases in NOE as CD4 titrated in

NMR-Based Screening in Drug Discovery

References

Reviews

Peng, J. W., J. Moore and N. Abdul-Manan (2004). "NMR experiments for lead generation in drug discovery." Progress in Nuclear Magnetic Resonance Spectroscopy 44(3-4): 225-256.

Lepre, C. A., J. M. Moore and J. W. Peng (2004). "Theory and applications of NMR-based screening in pharmaceutical research." Chemical Reviews 104(8): 3641-3675.

Meyer, B. and T. Peters (2003). "NMR Spectroscopy techniques for screening and identifying ligand binding to protein receptors." Angewandte Chemie-International Edition 42(8): 864-890.

Stockman, B. J. and C. Dalvit (2002). "NMR screening techniques in drug discovery and drug design." Progress in Nuclear Magnetic Resonance Spectroscopy 41(3-4): 187-231.

Hajduk, P. J., R. P. Meadows and S. W. Fesik (1999). “NMR-based screening in drug discovery.” Quarterly Reviews of Biophysics 32(3): 211-240.

Techniques

• SAR by NMR

Shuker, S. B., P. J. Hajduk, R. P. Meadows and S. W. Fesik (1996). "Discovering high-affinity ligands for proteins: SAR by NMR." Science 274(5292): 1531-4.

Hajduk, P. J., G. Sheppard, D. G. Nettesheim, E. T. Olejniczak, S. B. Shuker, R. P. Meadows, D. H. Steinman, G. M. Carrera, P. A. Marcotte,J. Severin, K. Walter, H. Smith, E. Gubbins, R. Simmer, T. F. Holzman,D. W. Morgan, S. K. Davidsen, J. B. Summers and S. W. Fesik (1997).“Discovery of Potent Nonpeptide Inhibitors of Stromelsin Using SAR byNMR.” Journal of the American Chemical Society 119: 5818-5827.

• STD NMR

Mayer, M. and B. Meyer (1999). “Characterization of ligand binding bysaturation transfer difference NMR spectroscopy.” Angewandte ChemieInternational Edition 38(12): 1784-1788.

• Relaxation Edited NMRHajduk, P. J., E. T. Olejniczak and S. W. Fesik (1997). “One-dimensionalrelaxation- and diffusion-edited NMR methods for screening compoundsthat bind to macromolecules.” Journal of the American Chemical Society119(50): 12257-12261.

• Diffusion Edited NMRBleicher, K., M. Lin, M. J. Shapiro and J. R. Wareing (1998). “DiffusionEdited NMR: Screening Compound Mixtures by Affinity NMR to DetectBinding Ligands to Vancomycin.” Journal of Organic Chemistry 63:8486-8490.

Techniques

• H2O-editingDalvit, C., P. Pevarello, M. Tato, M. Veronesi, A. Vulpetti and M.Sundstrom (2000). “Identification of compounds with binding affinity toproteins via magnetization transfer from bulk water.” Journal ofBiomolecular Nmr 18(1): 65-68.

NOE-based Methods• Transfer NOEMeyer, B., T. Weimar and T. Peters (1997). “Screening mixtures for biological activity by NMR.” European Journal of Biochemistry 246(3): 705-9.

• NOE pumping:Chen, A. and M. J. Shapiro (1998). “NOE pumping: A novel NMRtechnique for identification of compounds with binding affinity tomacromolecules.” Journal of the American Chemical Society 120(39):10258-10259.

NMR screening in Drug Discovery

Many therapies rely on the use of a drug that interacts with a cellular target and alters its activity. Drug discovery is the process of identifying and optimizing drug candidates and is usually a time consuming task that consists of several critical stages:

• target selection• lead generation• lead optimization• preclinical development

The lead generation step identifies starting compounds or “leads” with binding activity against the target and usually requires the screening of a very large number of compounds (>106).

It is important to note that most compounds in a library will not bind to the target and those that interact, bind only weakly (KD ~ uM-mM)

Compounds in the library that interact with the target are likely to bind only weakly (uM – mM). NMR is ideally suited for screening a library of compounds for weak to tight binding in a high throughput fashion and is utilized in one of the following modes:

• target or receptor-based screening - does ligand interact with target by following changes in the target chemical shifts. Observe and compare chemical shifts of target in the absence and presence of ligand (SAR by NMR)

• ligand-based screening – does ligand interact with target by following changes in ligand NMR parameters upon adding target:

• saturation transfer difference (STD)• relaxation editing• diffusion editing• hydration• NOE based methods

SAR by NMR

SAR by NMR is carried out in five main steps:1. Identification of ligands with high binding affinity from a library of

compounds by using 2D 1H-15N HSQC2. Optimization of ligands by chemical modification3. Identification of ligands binding in the presence of saturating amounts of

optimized ligands from step 2 by using 2D 1H-15N HSQC4. Optimization of ligands for the second site5. Tethering the two ligands from step 2 and step 4 in various positions and

checking again for binding

Shuker et al. (Science 274, 1531 (1996)) used SAR by NMR to screen 1000 substances for FK506-like binding activity. FK506 is an important drug that binds FKBP and suppresses rejection reactions after organ transplants but it has high toxicity. SAR by NMR yielded a tethered ligand with high affinity for FKBP (KD=19 nM). The complex was then subjected to a detailed structural analysis using NMR.

STD NMR – Saturation Transfer Difference

STD involves selectively saturating a resonance that belongs to the receptor (must find a region of the spectrum that contains only resonances from receptor such as 0 ppm to -1 ppm). If ligand binds, saturation will propagate from the selected receptor protons to other protons of receptor via spin diffusion and then the saturation is transferred to binding compounds by cross relaxation at the ligand-receptor interface. A control experiment is done by saturating a region of the spectrum that does not contain signal. The resulting difference spectrum yields only those resonances that have experienced saturation, namely those of the receptor and those of the compound that binds to the receptor. The receptor is typically present at very small concentrations so its resonances will not be visible and, if needed, can be eliminated by a relaxation filter.

Mayer and Meyer (Angewandte Chemie 38, 1784 (1999) used STD NMR to screen a library of carbohydrate molecules for binding activity towards a carbohydrate binding protein, wheat-germ agglutinin (WGA). STD can also be used for determining the binding epitope of the ligand, information that is of prime importance for the directed development of drugs.

Epitope Mapping Using STD NMR

STD NMR can be used to identify the ligand fragments or epitopes that are in direct contact with the receptor because these components receive the highest degree of to saturation.

Diffusion Edited Screening

A variety of NMR pulse sequences has been developed that allow the investigation of diffusion constants in solution. On the basis of such experiments it is possible to discriminate compounds in mixtures according to their diffusion properties. This so-called diffusion editing has been successfully applied to deconvolute compound mixtures, and to detect molecular association processes. For small- and intermediate-size molecules the approach works well, and in principle, diffusion editing is also applicable to the detection of binding of low-molecular-weight compounds to large protein receptors.

First, a PFG-STE or PFG-SE spectrum of the chemical mixture in the absence of the protein is recorded at a low gradient strength. Next, the same spectra for the chemical mixture in the presence of the protein are recorded at low and high gradient strengths and subtracted to produce a spectrum that contains only the signals of the compounds not interacting with the protein. The resulting subtracted spectrum is then subtracted from the spectrum of the chemical mixture recorded in the absence of the protein to obtain a spectrum that contains only the signals of the molecule that binds to the receptor.

Hajduk et al. (JACS 119, 12257 (1997)) used diffusion editing to demonstrate that 4-cyano-40-hydroxybiphenyl, which binds to stromelysin with a dissociation constant of 20 M, was easily identified from a mixture containing eight other non-binding compounds.

A comparison of methods for ligand-based and target-based NMR screening in drug discovery: