Probing and modulating membrane proteins

There are four research areas in the Department of Pharmaceutical Chemistry. Probing and modulating membrane proteins is a research challenge within protein and cellular engineering.

The challenge

Membrane proteins—in particular integral proteins that span cellular membranes—are encoded by about a quarter of the human genome. They play vital biological roles as channels, transporters, adhesion molecules, and receptors; relaying signals and transferring energy, molecules, and ions across membranes, including between the cell and its environment. Indeed, they are the most common drug targets.

But analyses of their membrane-embedded (transmembrane) helical domains to determine how they fold and assemble into their specific structures and how those structures—plus interactions between proteins or intra-protein domains—dictate function, lag behind studies of water-soluble proteins.

This disparity is partly due to their lower levels of expression, greater flexibility, and the need for complex model systems that replicate cell membranes—making experimental determination of their structures more challenging. (Membrane proteins comprise about one percent of the determined structures in the global Protein Data Bank).

In particular, engineered antibodies or other reagents can selectively bind water-soluble proteins (or water-soluble regions of membrane proteins), providing insights into their structure and function, plus, potentially, therapeutic inhibitors of protein activity. But there has been a lack of such probes to target and modulate proteins’ membrane-embedded domains.

Examples of our research and methods include

Developing peptides to bind transmembrane domains

Department scientists have computationally designed membrane-spanning helical peptides that recognize and bind to the membrane-embedded helices of transmembrane proteins with high sequence specificity.

The method, dubbed computed helical anti-membrane protein, or CHAMP, selects a backbone geometry to complement target helices based on structural databases of known helix pairs in transmembrane proteins. Then it further selects for the best adjusted fit (re-packing) between amino acid side chains at the interface of the CHAMP-generated and target helix. (In the membrane environment it is hypothesized that precise geometric complementarity is especially crucial for binding selectivity and affinity. But it should also be possible to extend this method to helix associations involving polar side chains, which have also been found to drive the association of model transmembrane peptides).

Close-up of computer-predicted interface between a CHAMP peptide and its target, the membrane-embedded helices of alphaIIbeta transmembrane integrin proteins. The surface of the protein domain is shown in red and blue. CHAMP backbone is gray with key positions designated for computational design shown in green.

Our applications of such protein engineering include

Targeting transmembrane domains of platelet receptors

The CHAMP method has been tested by targeting the very similar (homologous) transmembrane domains of two closely related types of membrane receptors in platelets, circulating cell fragments that play a key role in blood clotting. The targeted receptors, called integrins, have a large extracellular domain, transmembrane helices, and a short cytoplasmic domain. They induce platelets to stick to one another or to matrix proteins, thus contributing to clotting.

It was determined that the CHAMP peptides were able to:

Assume an alpha helical shape and insert themselves into and across phospholipid bilayers that comprise cell membranes, like those of human red blood cells such as platelets, without lysing them.
Selectively interact with their target integrins, even when they were outnumbered several hundred-fold by homologous isoforms.
Induce integrin activation and thus platelet and/or platelet-matrix adhesion in assays using mammalian cells—as well as in models such as detergent micelles.
Help confirm that, in their resting state, the heterodimer integrins’ transmembrane helices interact. It is when they are disassociated, as with competing CHAMP peptide binding, that integrins become active.

A schematic diagram of integrin regulation by CHAMP peptides. Integrins are heterodimer transmembrane receptor proteins endogenously activated by ADP (left). When inactive, their alpha and beta membrane-embedded domains interact (center). CHAMP peptides block this interaction in the membrane, activating the protein (right).

Such modulation of the transmembrane domains of integrins has potential therapeutic applications in safer reduction of pathological clotting leading to heart attack and stroke. It could also reduce scarring of blood vessels in the kidney (glomeruli) that can contribute to end-stage renal disease. Glomerulosclerosis can be caused by a type of integrin that upregulates collagen synthesis, thus its inhibition reduces glomerular injury.