Structural Biochemistry/Proteins/Integrin Proteins

General
Integrin proteins are receptor molecules that are adhesive to cells that mediate attachment between a cell and the tissues surrounding it, which include other cells or the extracellular matrix. Integrin proteins get their name from their ability to integrate the extracellular and intracellular environments by binding to ligands outside the cell and signaling molecules inside the cell. They also play important roles in the immune system, cell signaling, phagocytosis, cell migration, extracellular matrix assembly, and regulating the cell cycle. Integrin proteins are known to transmit information about how the cell is doing internally to and from the extracellular matrix. This allows the rapid response to changes in the external environment, which allows the maintenance of homeostasis. A good example of this is blood coagulation by platelets. Usually, receptors inform the cell of molecules near its environments so that the cell can initiate a response, integrin proteins perform this outside-in signaling but they can also perform inside-out signaling. Inside-out signaling is when they transduce information from the extracellular matrix to the cell and also reveals the status of the cell to the outside allowing quick responses to changes in environment. Integrins are usually found in an inactive state where they are not currently binding to receptors until needed. There are many types of integrin proteins, and many cells have multiple types on their surface. Integrin proteins can be found in just about any animal cell, and they have been exhaustively studied in humans especially.

Structure
Integrins are heterodimeric molecules that are associated noncovalently. 18 α subunits and 8 β subunits form 24 known αβ pairs in vertebrates. This diversity accounts for the diversity in ligand recognition (Figure 1), binding to cytoskeletal components and coupling to downstream signaling pathways. The β2 and β7 integrins are only expressed on leukocytes, whereas the β1 integrins are expressed on a large variety of cells throughout the body.

Priming, also known as inside-out signaling, dynamically regulates the adhesiveness of integrins by receiving stimuli from cell surface receptors which detect chemokines, cytokines, and foreign antigens which results in intracellular signals to be sent which will alter adhesiveness for extracellular ligands. Also, ligand binding transduces signals form the extracellular domain to the cytoplasm in the outside-in direction. These properties are the key to their proper function in the immune system.

Structure
Half of integrin α subunits include a region of 200 amino acids known as inserted (I) domains, or a von Willebrand factor A domain. The α I domain is the major ligand binding site in the integrins which contain this region. This domain was the first domain to be crystallized. The α I domain favors the dinucleotide binding with α-helices surrounding a central β-sheet. Beta strands and alpha helices usually alternate in secondary structure resulting in alpha helices wrapping around the domain in a counter clockwise order when viewed from the top face. The top face of the domain is defined by a divalent cation-biding site which physiologically binds Mg 2+. The Mg 2+ becomes ligated by 5 side chains located in three different loops. The first loop contains three coordinating residues in a signature sequence of I domains: Asp, Ser, Ser. The second loop donates a coordinating Thr residue and the third loop donates an Asp. This site is called the MIDAS (metal ion-dependent adhesion site).

Conformational Regulation of α I Domains
I domains have been crystallized in three conformations: Closed, Intermediate, Open. These demonstrate the coordination of the metal in MIDAS, the arrangement of the beta six and alpha seven loops, and the axial repositioning of the C-terminal alpha seven helix along the side of the I domain. Oxygen is donated to the primary and secondary coordination spheres surrounding the metal via the five residues and several water molecules. In the open conformation of MIDAS, two serines and one threonine occupy the primary coordination sphere and two aspartic residues occupy the secondary coordination sphere. The glutamic acid residue donates only negatively charged oxygen to the primary coordination sphere in the open conformation. A hypothesis exists which states that the strength of the metal-ligand bond is enhanced because of the lack of any charged group in the primary coordination sphere that is donated by the I domain. The closed conformation of the alpha I domain results in the threonine from the primary coordination sphere to switch with the aspartic acid residue from the secondary coordination sphere. The backbone and side chain rearrangements are followed by a 2.3 angstrom sideways movement of the metal ion away from the threonine and closer to the aspartic acid which is on the opposite side of the coordination cell. The closed and open structures follow the idea that an energetically favorable MIDAS requires at least one coordination to a negatively charged oxygen. When there is no ligand, a pseudoligand, and the remainder of the integrin ectodomain crystallize in the closed conformation. This closed conformation is the low energy conformation, which is verified computationally. However, through an engineered disulfide bond, the alpha L I domain is crystallized in the open confirmation and is stable in the absence of ligand-mimetic lattice contact. As a result, interactions with other integrin domains might be capable of stabilizing an unliganded I domain in the open conformation and priming it for ligand binding.

Allosteric α I Domain Inhibitors
Small allosteric inhibitors provide further support for the role of the alpha seven helix in alpha I domain regulation. Alpha I allosteric antagonists are a class of small molecule inhibitors which binds underneath the C-terminal α-helix of the αL I domain. These antagonists stabilize the closed conformation of the I domain by stopping the downward axial movement of the alpha seven helix and preventing the MIDAS rearrangements necessary for efficient ligand binding. The action of these antagonists is confirmed by finding that a mutant alpha L I domain which stabilizes the open conformation of the C-terminal alpha seven-helix with an engineered disulfide bond is resistant to inhibition by alpha I allosteric antagonists.

Integrin Activation
Chemokine in leukocytes, thrombin in platelets, and T-cell receptor engagement in T-cells are certain examples that cause integrin activation. Once activated, there is an increase in calcium and diacyl glycerol (DAG) levels which causes activation of a guanine nucleotide exchange factor (GEF). Rap1 is then activated, causing GDP/GTP exchange. Once Rap1 has been activated, it works together with RIAM, an interacting adapter molecule which helps connect the membrane targeting sequences in Rap1 to talin, and talin is then bound to the plasma membrane where it causes the β-TM helix to tilt, leading to full integrin activation. A complex formed by the β-tail, talin and the cell membrane is an important part of inside-out integrin activation in the cell, and thus the formation of this complex is regulated to ensure that integrin activation is under control. Β-tail and talin affinity is also controlled by environmental conditions, as well as competition from other proteins that compete for interaction with the β-integrin tail. A certain type of proteins called kindlins also aid in talin activation of integrins. Kindlins contain FERM domains that are similar to talins with their N-terminal Fo domain and a large flexible F1 loop. Unlike talin which binds to the first NpxY motif in β-integrin tails, Kindlins bind to the second motif. However, Kindlin has also been known to be able to also inhibit integrins as well.