William F. DeGrado

How calmodulin binds its targets : sequence independent recognition of amphiphilic α-helices

Calmodulin (CaM) is a protein capable of recognizing positively charged, amphiphilic alpha-helical peptides independent of their precise amino acid sequences; this structural feature has also been found in many CaM-binding proteins. Recent work involving crystallography and site-directed mutagenesis of CaM along with studies of photoreactive and fluorescent CaM-binding peptides have helped define how calmodulin interacts with amphiphilic helices.

Structural basis for the function and inhibition of an influenza virus proton channel

The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza drugs amantadine and rimantadine; recently, resistance to these drugs in humans, birds and pigs has reached more than 90% (ref. 1). Here we describe the crystal structure of the transmembrane-spanning region of the homotetrameric protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Binding of amantadine physically occludes the pore, and might also perturb the pKa of the critical His residue. The structure provides a starting point for solving the problem of resistance to M2-channel blockers.

Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers.

The M2 protein of influenza A virus is a membrane-spanning tetrameric proton channel targeted by the antiviral drugs amantadine and rimantadine. Resistance to these drugs has compromised their effectiveness against many influenza strains, including pandemic H1N1. A recent crystal structure of M2(22–46) showed electron densities attributed to a single amantadine in the amino-terminal half of the pore, indicating a physical occlusion mechanism for inhibition. However, a solution NMR structure of M2(18–60) showed four rimantadines bound to the carboxy-terminal lipid-facing surface of the helices, suggesting an allosteric mechanism. Here we show by solid-state NMR spectroscopy that two amantadine-binding sites exist in M2 in phospholipid bilayers. The high-affinity site, occupied by a single amantadine, is located in the N-terminal channel lumen, surrounded by residues mutated in amantadine-resistant viruses. Quantification of the protein–amantadine distances resulted in a 0.3 Å-resolution structure of the high-affinity binding site. The second, low-affinity, site was observed on the C-terminal protein surface, but only when the drug reaches high concentrations in the bilayer. The orientation and dynamics of the drug are distinct in the two sites, as shown by 2H NMR. These results indicate that amantadine physically occludes the M2 channel, thus paving the way for developing new antiviral drugs against influenza viruses. The study demonstrates the ability of solid-state NMR to elucidate small-molecule interactions with membrane proteins and determine high-resolution structures of their complexes.

Protein Design: A Hierarchic Approach

The de novo design of peptides and proteins has recently emerged as an approach for investigating protein structure and function. Designed, helical peptides provide model systems for dissecting and quantifying the multiple interactions that stabilize secondary structure formation. De novo design is also useful for exploring the features that specify the stoichiometry and stability of α-helical coiled coils and for defining the requirements for folding into structures that resemble native, functional proteins. The design process often occurs in a series of discrete steps. Such steps reflect the hierarchy of forces required for stabilizing tertiary structures, beginning with hydrophobic forces and adding more specific interactions as required to achieve a unique, functional protein.

Asparagine-mediated self-association of a model transmembrane helix.

In membrane proteins, the extent to which polarity, hydrogen bonding, and van der Waals packing interactions of the buried, internal residues direct protein folding and association of transmembrane segments is poorly understood. The energetics associated with these various interactions should differ substantially between membrane versus water-soluble proteins. To help evaluate these energetics, we have altered a water-soluble, two-stranded coiled-coil peptide to render its sequence soluble in membranes. The membrane-soluble peptide associates in a monomer-dimer-trimer equilibrium, in which the trimer predominates at the highest peptide/detergent ratios. The oligomers are stabilized by a buried Asn side chain. Mutation of this Asn to Val essentially eliminates oligomerization of the membrane-soluble peptide. Thus, within a membrane-like environment, interactions involving a polar Asn side chain provide a strong thermodynamic driving force for membrane helix association.

De novo design of biomimetic antimicrobial polymers

The design of polymers and oligomers that mimic the complex structures and remarkable biological properties of proteins is an important endeavor with both fundamental and practical implications. Recently, a number of nonnatural peptides with designed sequences have been elaborated to provide biologically active structures; in particular, facially amphiphilic peptides built from β-amino acids have been shown to mimic both the structures as well as the biological function of natural antimicrobial peptides such as magainins and cecropins. However, these natural peptides as well as their β-peptide analogues are expensive to prepare and difficult to produce on a large scale, limiting their potential use to certain pharmaceutical applications. We therefore have designed a series of facially amphiphilic arylamide polymers that capture the physical and biological properties of this class of antimicrobial peptides, but are easy to prepare from inexpensive monomers. The design process was aided by molecular calculations with density functional theory-computed torsional potentials. This new class of amphiphilic polymers may be applied in situations where inexpensive antimicrobial agents are required.

Design of peptides and proteins.

Publisher Summary This chapter focuses on contemporary approaches to the design of structurally defined peptides and proteins with special emphasis being given to the ways in which this approach has elucidated the structural basis for the function of a variety of natural molecules. When in their proper biological milieu, peptides and proteins assume fairly well-defined conformations, which are responsible for their biological and physical properties. The mechanisms by which these molecules adopt defined three-dimensional structures depend on their size and chemical compositions. There are a variety of strategies and approaches for protein and peptide design. Those adopted for a given problem will depend on the size and molecular characteristics of the desired target molecule. If one wishes to create a model for a small peptide (e.g., the pentapeptide hormone enkephalin), then it might be feasible to design an analog of this molecule that is locked into a biologically active conformation. The design of proteins that adopt predetermined three-dimensional structures is perhaps the most ambitious goal, and one that has been attacked only recently. The design of conformationally constrained peptides is a powerful method for elucidating the biologically active conformation of a peptide when it is bound to its receptor. The design of medium-sized peptides is beset with many of the same problems encountered in the design of small peptides. Medium-sized peptides are also very flexible and tend to lack defined conformations at room temperature in aqueous solutions.

Computational Design of Peptides That Target Transmembrane Helices

A variety of methods exist for the design or selection of antibodies and other proteins that recognize the water-soluble regions of proteins; however, companion methods for targeting transmembrane (TM) regions are not available. Here, we describe a method for the computational design of peptides that target TM helices in a sequence-specific manner. To illustrate the method, peptides were designed that specifically recognize the TM helices of two closely related integrins (αIIbβ3 and αvβ3) in micelles, bacterial membranes, and mammalian cells. These data show that sequence-specific recognition of helices in TM proteins can be achieved through optimization of the geometric complementarity of the target-host complex.

Helix-packing motifs in membrane proteins

The fold of a helical membrane protein is largely determined by interactions between membrane-imbedded helices. To elucidate recurring helix–helix interaction motifs, we dissected the crystallographic structures of membrane proteins into a library of interacting helical pairs. The pairs were clustered according to their three-dimensional similarity (rmsd ≤1.5 Å), allowing 90% of the library to be assigned to clusters consisting of at least five members. Surprisingly, three quarters of the helical pairs belong to one of five tightly clustered motifs whose structural features can be understood in terms of simple principles of helix–helix packing. Thus, the universe of common transmembrane helix-pairing motifs is relatively simple. The largest cluster, which comprises 29% of the library members, consists of an antiparallel motif with left-handed packing angles, and it is frequently stabilized by packing of small side chains occurring every seven residues in the sequence. Right-handed parallel and antiparallel structures show a similar tendency to segregate small residues to the helix–helix interface but spaced at four-residue intervals. Position-specific sequence propensities were derived for the most populated motifs. These structural and sequential motifs should be quite useful for the design and structural prediction of membrane proteins.

De novo design and in vivo activity of conformationally restrained antimicrobial arylamide foldamers

The emergence of drug-resistant bacteria has compromised the use of many conventional antibiotics, leading to heightened interest in a variety of antimicrobial peptides. Although these peptides have attractive potential as antibiotics, their size, stability, tissue distribution, and toxicity have hampered attempts to harness these capabilities. To address such issues, we have developed small (molecular mass <1,000 Da) arylamide foldamers that mimic antimicrobial peptides. Hydrogen-bonded restraints in the arylamide template rigidify the conformation via hydrogen bond formation and increase activity toward Staphylococcus aureus and Escherichia coli. The designed foldamers are highly active against S. aureus in an animal model. These results demonstrate the application of foldamer templates as therapeutics.