How does the clustering of phosphatidylinositol phosphates assist in pleckstrin homology domain binding of membranes?

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The cells which make up our bodies consist at the simplest level of a lipid membrane containing a water-filled space controlled from the centre by the cell nucleus (where DNA is stored and which has its own secondary membrane for protection). Cells have to signal to one another and possess systems of communication in order to regulate the basic processes of life: DNA replication, cell division, cell growth and, by cell-cell interaction, the formation of tissues and organs. The major signalling routes employed are based on proteins which bind to one another and to other molecules like DNA and lipids. This means that which proteins a cell expresses and at what levels will determine how it behaves and the interactions it enjoys with its surroundings.To enable the efficient evolution of cell signalling natural selection relies on protein "modules" or "domains". Individual modules crop up in multiple forms, and each kind shares a common basic structure adapted in each case to a different activity. This means that rather than interactions between two molecules having evolved repeatedly from scratch, over the course of evolution existing interaction interfaces have been adapted for new roles.This grant proposal concerns one important module type, the pleckstrin homology (or PH) domain. PH domains are associated with protein-membrane interactions but also get involved in protein-protein interfaces. When binding membranes, PH domains often interact with special lipid molecules which have been modified by the chemical addition of phosphate groups, in particular lipids called phosphatidyl inositol phosphates (PIPs). Although some PH domains have been shown to bind tightly and very specifically to one kind of PIP (they have high affinity), others seem to have a low affinity - maybe too low to be relevant in the complex environment of a cell - and not to be very choosy about binding partners. We recently found that one PH domain seems to bind not to a single PIP but to several PIPs clustered together and that this increases the tightness of its binding about 300x. We are proposing that this mechanism whereby a PH domain relies on strength in numbers to target specific membranes could be relevant to a number of other PH domains whose measured affinity for their putative PIP target is low.Our starting point will be to look at the PH domains of three related human proteins, called kindlin-1, kindlin-2 and kindlin-3. Having looked at kindlin-3 already, we want to see if the other two members of this small protein family have similar properties and recognise clusters of PIPs with increased affinity. All the data we have collected to date suggest that they probably will.Kindlins are an interesting group of proteins to study because they have very imporant roles at several different points in the cell, and so understanding how it is that they bind to one membrane in one part of the cell or move to another part of the cell is something that matters very much. For example, the kindlins are well known to activate mechanisms by which cells stick to one another, but they also control things like growth signals and move to the cell nucleus to take up completely different activities, controlling gene expression.We will compare the power of PIP clustering to enhance kindlin PH domain binding, to other proteins which we already know bind tightly to a single PIP molecule. Then we will test whether more proteins considered to have low PIP affinity make use of "strength in numbers" to tighten their binding. The proteins we are going to study are known to be important in disease, including a large number of different cancers, and also heart disease and stroke. This means that investigating the possibility of a new kind of mechanism which underlies their biological effect is something that can help us understand better factors influencing the roles they play - not only in normal cell function but also when things go wrong.