William C. Wimley

HYDROPHOBIC INTERACTIONS OF PEPTIDES WITH MEMBRANE INTERFACES

The thermodynamic principles underlying the structural stability of membrane proteins are difficult to obtain directly from whole proteins because of intractable problems related to insolubility in the aqueous phase and extreme stability in the membrane phase. The principles must therefore be surmised from studies of the interactions of small peptides with lipid bilayers. This review is concerned with the hydrophobic interactions of such peptides with the interfacial regions of lipid bilayers. We first develop a general framework for thinking about the thermodynamics of membrane protein stability that centers on interfacial interactions and review the structural and chemical evidence that supports this interface-centered point of view. We then describe an experimentally determined whole-residue interfacial hydrophobicity scale that reveals the central role of the peptide bond in partitioning and folding. Finally, we consider the complexity and diversity of interfacial interactions revealed by differences between side-chain hydrophobicities determined using different classes of peptides.

Structure, function, and membrane integration of defensins

Defensins comprise a structural class of small cationic peptides that exert broad-spectrum antimicrobial activities through membrane permeabilization. Their predominantly beta-sheet structure, stabilized by three disulfide bonds, distinguishes them from other antimicrobial peptides which typically form amphiphilic helices. Defensins bind to membranes electrostatically and subsequently form apparently multimeric pores. Recent structural and biophysical studies are beginning to provide insights into the process of permeabilization.

The versatile β-barrel membrane protein

Abstract The β-barrel membrane protein is found in the outer membranes of bacteria, mitochondria and chloroplasts. Approximately 2–3% of the genes in Gram-negative bacterial genomes encode β-barrels. Whereas there are fewer than 20 known three-dimensional β-barrel structures, genomic databases currently contain thousands of β-barrels belonging to dozens of families. New research is revealing the variety of β-barrel structures and the variety of functions performed by these versatile proteins.

Antimicrobial Peptides: successes, challenges and unanswered questions

Multidrug antibiotic resistance is an increasingly serious public health problem worldwide. Thus, there is a significant and urgent need for the development of new classes of antibiotics that do not induce resistance. To develop such antimicrobial compounds, we must look toward agents with novel mechanisms of action. Membrane-permeabilizing antimicrobial peptides (AMPs) are good candidates because they act without high specificity toward a protein target, which reduces the likelihood of induced resistance. Understanding the mechanism of membrane permeabilization is crucial for the development of AMPs into useful antimicrobial agents. Various models, some phenomenological and others more quantitative or semimolecular, have been proposed to explain the action of AMPs. While these models explain many aspects of AMP action, none of the models captures all of the experimental observations, and significant questions remain unanswered. Here, we discuss the state of the field and pose some questions that, if answered, could speed the discovery of clinically useful peptide antibiotics.

Toward genomic identification of β-barrel membrane proteins: Composition and architecture of known structures

The amino acid composition and architecture of all β‐barrel membrane proteins of known three‐dimensional structure have been examined to generate information that will be useful in identifying β‐barrels in genome databases. The database consists of 15 nonredundant structures, including several novel, recent structures. Known structures include monomeric, dimeric, and trimeric β‐barrels with between 8 and 22 membrane‐spanning β‐strands each. For this analysis the membrane‐interacting surfaces of the β‐barrels were identified with an experimentally derived, whole‐residue hydrophobicity scale, and then the barrels were aligned normal to the bilayer and the position of the bilayer midplane was determined for each protein from the hydrophobicity profile. The abundance of each amino acid, relative to the genomic abundance, was calculated for the barrel exterior and interior. The architecture and diversity of known β‐barrels was also examined. For example, the distribution of rise‐per‐residue values perpendicular to the bilayer plane was found to be 2.7 ± 0.25 Å per residue, or about 10 ± 1 residues across the membrane. Also, as noted by other authors, nearly every known membrane‐spanning β‐barrel strand was found to have a short loop of seven residues or less connecting it to at least one adjacent strand. Using this information we have begun to generate rapid screening algorithms for the identification of β‐barrel membrane proteins in genomic databases. Application of one algorithm to the genomes of Escherichia coli and Pseudomonas aeruginosa confirms its ability to identify β‐barrels, and reveals dozens of unidentified open reading frames that potentially code for β‐barrel outer membrane proteins.

Mechanism Matters: A Taxonomy of Cell Penetrating Peptides.

The permeability barrier imposed by cellular membranes limits the access of exogenous compounds to the interior of cells. Researchers and patients alike would benefit from efficient methods for intracellular delivery of a wide range of membrane-impermeant molecules, including biochemically active small molecules, imaging agents, peptides, peptide nucleic acids, proteins, RNA, DNA, and nanoparticles. There has been a sustained effort to exploit cell penetrating peptides (CPPs) for the delivery of such useful cargoes in vitro and in vivo because of their biocompatibility, ease of synthesis, and controllable physical chemistry. Here, we discuss the many mechanisms by which CPPs can function, and describe a taxonomy of mechanisms that could be help organize future efforts in the field.

Protein folding in membranes: determining energetics of peptide-bilayer interactions.

Although the problem of the folding of soluble proteins continues to resist solution, we at least have a strong understanding of the general thermodynamic principles1,2 and have available a wealth of thermodynamic data.3-5 The study of membrane protein folding and stability is much less advanced: Some general principles are emerging,6-9 but the amount of thermodynamic data available remains quite limited. The energetics of the partitioning of peptides into membranes constitutes one especially important class of data. We will demonstrate how such data can be used for clarifying the folding of peptides and small proteins in membranes and then describe the principles and methods used for determining the energetics of the partitioning of peptides into bilayer membranes.

The ERBB4/HER4 Intracellular Domain 4ICD Is a BH3-Only Protein Promoting Apoptosis of Breast Cancer Cells

ERBB4/HER4 (referred to here as ERBB4) is a unique member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In contrast to the other three members of the EGFR family (i.e., EGFR, ERBB2/HER2/NEU, and ERBB3), which are associated with aggressive forms of human cancers, ERBB4 expression seems to be selectively lost in tumors with aggressive phenotypes. Consistent with this observation, we show that ERBB4 induces apoptosis when reintroduced into breast cancer cell lines or when endogenous ERBB4 is activated by a ligand. We further show that ligand activation and subsequent proteolytic processing of endogenous ERBB4 results in mitochondrial accumulation of the ERBB4 intracellular domain (4ICD) and cytochrome c efflux, the essential and committed step of mitochondrial regulated apoptosis. Our results indicate that 4ICD is functionally similar to BH3-only proteins, proapoptotic members of the BCL-2 family required for initiation of mitochondrial dysfunction through activation of the proapoptotic multi-BH domain proteins BAX/BAK. Similar to other BH3-only proteins, 4ICD cell-killing activity requires an intact BH3 domain and 4ICD interaction with the antiapoptotic protein BCL-2, suppressed 4ICD-induced apoptosis. Unique among BH3-only proteins, however, is the essential requirement of BAK but not BAX to transmit the 4ICD apoptotic signal. Clinically, cytosolic but not membrane ERBB4/4ICD expression in primary human breast tumors was associated with tumor apoptosis, providing a mechanistic explanation for the loss of ERBB4 expression during tumor progression. Thus, we propose that ligand-induced mitochondrial accumulation of 4ICD represents a unique mechanism of action for transmembrane receptors, directly coupling a cell surface signal to the tumor cell mitochondrial apoptotic pathway.

Peptides in lipid bilayers: structural and thermodynamic basis for partitioning and folding

Abstract The lipid bilayer is commonly, but incorrectly, viewed as a slab of bulk hydrocarbon liquid bounded by thin polar regions. Recent experimental and theoretical advances reveal the true complexities of the bilayer and its interactions with peptides. These are reviewed in the context of five fundamental questions that must be addressed in order to arrive at a structural and thermodynamic framework that is useful for studies of a broad array of biological processes.

Transmembrane helix dimerization: beyond the search for sequence motifs.

Studies of the dimerization of transmembrane (TM) helices have been ongoing for many years now, and have provided clues to the fundamental principles behind membrane protein (MP) folding. Our understanding of TM helix dimerization has been dominated by the idea that sequence motifs, simple recognizable amino acid sequences that drive lateral interaction, can be used to explain and predict the lateral interactions between TM helices in membrane proteins. But as more and more unique interacting helices are characterized, it is becoming clear that the sequence motif paradigm is incomplete. Experimental evidence suggests that the search for sequence motifs, as mediators of TM helix dimerization, cannot solve the membrane protein folding problem alone. Here we review the current understanding in the field, as it has evolved from the paradigm of sequence motifs into a view in which the interactions between TM helices are much more complex. This article is part of a Special Issue entitled: Membrane protein structure and function.