Created by: Monica Nazir

The VPS9-domain ankyrin repeated protein, also known as VARP, (PDB ID: 4CYM) is a mammalian protein involved in complex features of cellular processes, specifically as a transporter protein. It is present mostly in multicellular organisms, but is found in some unicellular systems, making it a model protein for research. It is a Rab32/38 effector (5), involved in SNARE complexes that further regulate movement of cargo within the cell. This family of Rab effector proteins has been found to participate in recruitment mechanisms within a cell. A multi-domain particle with 1,050 residues, it expresses functionally important segments and locations of affinity for binding to other complexes, including the P loop and the ankyrin repeated domains, discussed in more detail below. Of special note is its interaction with the R-SNARE molecules VAMP7 and VSP29. VARP associates with several other protein complexes to carry out biologically significant tasks. Among these tasks is its role in localizing GLUT1 transporters from the cell’s endosome to the extracellular region of the membrane.  It is also involved in cells fusion events, bringing molecules into and out of the cell, and more specifically, fusion events regarding the endosome and lysosome system. To learn more about this protein, it is supplementary to use comparison structures that are similar yet distinct from VARP. Servers such as the Dali server and PSI-BLAST can serve this purpose, each comparing different aspects of protein structure and dynamics. 

GLUT1 transporters must be recruited from the endosomal region to the cell membrane and back to the cell interior. GLUT1 transport is significant to the cell’s energy reserve and metabolism, required for its utility. It is a retromer-dependent molecule. VARP interacts with the retromer that carries GLUT1. It binds to VSP29 (5). This family of GTPases that interact with Rab effectors are part of the network that manages transport within the cell. This factor makes VARP crucial for study regarding these movement-facilitating networks. VARP follows the specific path of the Rab32/38 effector to target endocytic organelles, as well as melanosomes, for the GLUT1 reception. VARP also forms complexes with individual Rab32 and Rab38 effectors. This is done through the first ankyrin repeating domain found between residues 451-640. This is a GTP dependent binding, enabling the complex for GLUT1 networking. As regulated GLUT1 finishes one phase of its intracellular interception, VSP29 becomes degraded, as was observed in HeLe cells (1). This acts as a control regulator of VARP and its associated complexes.  

In addition to its role within the GLUT1 import network, VARP is linked to Rab21 GEF (guanine nucleotide exchange factor) activity. While at the same time associating with an R-SNARE molecule, VAMP7 facilitates the movement. The binding occurs after VARP arrives in one of its conformation states, a closed state. One of its long domain chains associates with VAMP7. This SNARE molecule is important for other functions of the cell as well, but VARP highlights and aids in the process of fusion events of vesicles into and out of the cell. In some cases VARP is found to assist in delivering vesicles of this and other SNARE compounds to the cell’s external side (4). This implicates significance for biological study because of the ability to manipulate this process to promote neurite outgrowth. However, this remains a vague yet ever-growing field. 

VARP has a molecular weight of 144.9 kDa (5) and an isoelectric point of 6.08, obtained from the ExPASy server (11). It’s composed of six main subunit chains. It contains two associated ligands. The primary one is GCP (a phosphoemethylphosphonic acid.) This structure is for the purpose of inducing crystallization within the protein. The second associated ligand for VARP is magnesium ion. This serves the same purpose of inducing crystallization within the protein. The ligands associate with all three chains at different times. The secondary structures involved include both alpha helices and beta sheets, and they stabilize the protein in the different pre and post complex binding conformations. Four of these alpha helices are found at the VARP/Rab binding region.  VARP is a tetramer; each monomer is observably stabilized by hydrogen bonding. Further stabilizing is the burial of around 6,400 Angstroms (5) of the tetramer. This minimizes the surface area interaction with the solvent. 

In total, there are eight ankyrin repeats (5). At the ankyrin repeat domains there is a curved stack (composed of beta sheets) with respective concave and convex sides. Rab effector molecules bind to the convex side of this associative curve. The concave side remains free, allowing for the binding of other molecules that may associate with the protein. When associated through its monomer with Rab23 for transport facilitation, the heterodimer formed associates with another to form firmer homodimers (5). 

Of note are two cysteine dense segments within VARP. These occur at residue segments 410-450 and 692-730. The repeating pattern is CHPLCXCXXC, X being any amino acid. This sequence is indicative of the associative presence of a metal with the protein. Although it is yet to be confirmed, it is postulated that the metal is zinc, making VARP a “double Zn-finger” containing protein (7.) This cysteine rich segment has high potential as a metal ion-binding site.A P loop exists at residue segment 33-41. The function of this loop is to stabilize the Mg2+ coordinated triphosphate section of the ligand that binds to the protein. Switch I exists at residues 48-61. This activating switch causes the protein to fold over a nucleotide molecule, stabilizing the gamma phosphate of the GppCp complex. Switch II (5) at residue segment 84-97 aids in stabilizing this complex formation. Leu-51, Ser-46, and Asn-143 bind VAMP7. Asp-675, Asp-679, and Asp 681 are part of the VAMP7/VARP binding complex as well as an ankyrin-repeating domain. 

     VARP undergoes conformational change when binding with the Rab effector VAMP7. The long chain of VARP compresses, inhibiting VAMP7 from forming a complex SNARE structure. This closed conformation acts as a form of regulatory machinery for the protein. This is the only major known conformational change VARP is known to undergo. Studies regarding the possibility of conformational changes resulting from the binding of a metal ion to the cysteine rich arm are being conducted using mass spectrometry as well as X-ray crystallography (4).

   Further study of this protein was attained through comparison to other unique proteins utilizing the Dali and PSI-BLAST servers. These yielded unique scores that are indicative of the sequential and even structural parallelism among the proteins. The PSI-BLAST server is used to find proteins with similar primary structure to a protein subject. It searches for sequence similarities. This program is efficient to use, as it utilizes a combination of both statistical data and algorithms, searching for gapped alignments between the sequences. The protein for comparison obtained from this server is Ras-related protein Rab-7a(PDB ID 3LAW.) The E value is the measure of similarity used in this server. An E value of less than 0.05 is significant for the protein. This protein is similar both structurally and functionally.  The comparison protein’s has an E value of 7x10-80.  This protein is also a transport protein, specifically functioning to promote proton transport in Homo sapiens (2.) This protein is a 2.8 Å crystal structure. It is a five domain, multi-domain protein. It contains both alpha and beta sheets, similar to VARP. This is functionally a GTP-dependent molecule, requiring the cleavage of a phosphate. Ras-related Rab-7a has two obsesrved ligands, GCP and magnesium ion, as observed for VARP. Similarly, they play the same role of aiding in crystallization of the protein. 

Another server used is the Dali server. Its purpose is to compare proteins based on their tertiary structures. This is attained by comparing the intramolecular distances of the given protein to the proteins within the database. A Z score is the measure used to compare the similarity of the structures. A Z score above 2 indicated suffient difference from the protein of interest for comparison. This protein, GTP-binding protein Rheb (PDB ID 3SEA), had a Z score of 22.7, indicating close relation to VARP. This protein is a hydrolase, composed of two chains that involve alpha helices and beta sheet associations. The length of the protein is about 167 Angstroms, so it is relatively smaller than VARP. It is found in Homo sapiens. 

The structurally significant unit of this protein is a G-domain where a nucleotide-binding pocket exists, allowing association with other Rab effectors and GEF factors. This protein interacts with four ligands.  Acetate ion ligand maintains shape and conformation. Gaunosine-5’-diphosphate also contributes to shape and conformation maintenance. Magnesium ion and GCP are associated ligands with this protein as with VARP, inducing crystallization. The metal ions involved are the two metal ligands this protein associates with. Functionally, this protein is important because it targets rapamycin, activating this mTOR within mammals. Asn-164 is where GTP hydrolysis occurs, involving the tuberous screlosis complex. Gln-64 is a catalytic regulating residue within its position on the second chain.