MSOE Center for BioMolecular Modeling
 

Cdc42 Interacting Protein 4

Description

This tutorial will show the structure and the function of Cdc42 Interacting Protein 4 (CIP4). CIP4 is a homodimeric protein with 3 major domains: F-BAR, SH3, and HR1. Through multerimerzation, CIP4 functions in endocytosis, where it helps to create elongated, tubular vesicles. Recall that CIP4 was included in the clickable cellular landscape of endocytosis (Figure 1).


Figure 1. Cellular landscape of endocytosis. Multiple CIP4's are shown as the pink proteins located just below the clathrin cage. Painting by David Goodsell.

Throughout the tutorial, you can view the F-BAR dimer of CIP4 to the right. It is important to remember that you are only viewing the F-BAR domain dimer of CIP4 and not a dimer of the entire protein. You can rotate the molecule by clicking and dragging it. Using your scroll wheel will zoom in and out on the molecule.

Section 1: Primary to Tertiary Structure

Learning Objectives:
  1. Explain hierarchy of protein structure and common features of each level of structure
  2. Understand how different experimental methods can be used to determine protein structure and the limitations of these methods

This section will focus on the structure of the CIP4 monomer. CIP4's primary structure consists of 545 amino acids1. The primary structure contains the linear amino acid sequence of CIP4's three main domains: F-BAR, HR1, and SH3.

Click Here When clicked, this will show a single F-BAR domain.

CIP4's secondary structure includes both of the major protein secondary structure motifs, the alpha helix and the beta sheet. The F-BAR and HR1 domains are primarily alpha helices and the SH3 domain consists of primarily beta sheets. However, a large amount of CIP4's secondary structure is unknown, because it is unable to be determined with the technology currently available (Figure 2). From the secondary structure of CIP4, the tertiary structure forms alpha helical bundles and a beta barrel. The F-BAR domain has three long alpha helices connected with two turning loops, forming an alpha helical bundle. The HR1 domain has two alpha helices connected by a short loop, resulting in adjacent alpha-helixes. The SH3 domain's beta sheets form a single beta barrel. Because the secondary structure between the domains of CIP4 is unknown, the tertiary structure is also unknown (Figure 2). The structure of the F-BAR domain was determined through crystallization and X-ray diffraction, while the HR1 and SH3 domains were determined using a technique called solution nuclear magnetic resonance (NMR).2, 3, 4.

Figure 2. Monomer of CIP4 showing the secondary and tertiary structure of the F-BAR, HR1, and SH3 domains. The structure of the protein in between these domains has not yet been determined and is therefore represented as an undefined linker region2, 3, 4.

While this is the structure of a single monomer, it is not a functional unit. To become a fully functional protein, CIP4 requires quaternary structure.

Section 2: Quaternary Structure

Learning Objectives:

  1. Understand that some proteins require a fourth level of structure (quaternary) to become fully functional
  2. Explain why protein folding can be driven by burial of hydrophobic amino acids and functional group interactions

The quaternary structure of CIP4 is composed of a homodimer (Figure 3).

Figure 3. Representation of the CIP4 homodimer. One monomer is shown in green and the other monomer in purple.2, 3, 4.

The dimer contact surface of the F-BAR domain's three helical bundles are rich in hydrophobic amino acids while the outer side is rich in hydrophilic amino acids5. This causes the F-BAR domain to adapt a banana-shaped structure.

Click Here When clicked, this will highlight the hydrophobic residues in yellow and the hydrophilic residues in blue. This spacefill representation helps highlight the amino acid sidechains. Be sure to click and rotate the molecule to compare the two surfaces.

Protein quaternary assembly is largely driven by the need to bury hydrophobic regions from the cytoplasm. To bury the hydrophobic region of the F-BAR domain on the monomer, two monomers come together at these regions to form a dimer. Forming the dimer is favorable because burying these large hydrophobic regions in the interior dimer contact surface creates a highly stable protein5.

Click Here When clicked, this will show the F-BAR domain in its dimeric form.

CIP4's structure not only allows it to dimerize but also multimerize, which allows it to function in endocytosis.

Section 3: CIP4 Dimers Work Together In Endocytosis

Learning Objectives:

  1. Explain why in order to perform some functions, proteins require multiple proteins to come together to form a complex
  2. State different bonds and interactions proteins may use to interact with each other
  3. State experimental methods used to determine what amino acids are necessary in cellular processes

The F-BAR dimers of multiple CIP4 proteins multimerize into a helical coat that surrounds the invaginating tubular vesicle during endocytosis (Figure 4).

Figure 4. Illustration of CIP4's F-BAR dimer creating a helical coat around the endocytosing vesicle2. This will cause an otherwise spherical vesicle to elongate. CIP4 is always on the inside of the cell (cytoplasmic location). Refer to figure 1, which also shows the CIP4 helical coat around an endocytosing vesicle.

This coat is held together by tip-to-tip and lateral interactions between F-BAR dimers. Most importantly for the lateral interactions, directly opposing phenylalanine 276 (F276) residues on the F-BAR dimers create hydrophobic interactions that help hold the helical coat together. This phenylalanine, located on the lateral side of the F-BAR domain, is required for the dimers to laterally interact with each other. This was demonstrated by one study that showed mutation of this phenylalanine to an aspartate inhibited CIP4 from binding the plasma membrane and therefore rendered it incapable of inducing endocytic tubulation in cells6.

Click Here When clicked, this will highlight phenylalanine 276 in yellow.

Other lateral interactions include ionic interactions between positively charged residues, lysine 66 and 273 (K66 and K273), in one dimer and negatively charged residues, glutamate 285 (E285) and aspartate 286 (D286), in the other. Mutation of these amino acids also decreased the ability of CIP4 to tubulate membranes6.

Click Here When clicked, this will highlight lysines 66 and 273 in red and glutamate 285 and aspartate 286 in blue.

All of these amino acids involved in lateral interactions are highly conserved throughout CIP4 family members. Based on these lateral interactions, neighboring dimers should overlap by about half of their length, like partially interlaced fingers6 (Figure 5).

Figure 5. This representation shows interactions between four neighboring F-BAR dimers. The labeled amino acids are conserved and contribute to the tip-to-tip and lateral interactions6.

Lysine 166 (K166) is the most important amino acid in the tip-to-tip interactions between F-BAR dimers. It forms hydrogen bonds with three main chain carbonyl groups of the adjacent dimer (Figure 5). The mutation of this amino acid to alanine potently compromised tube formation, showing its importance in forming the helical coat2.

Click Here When clicked, this will highlight lysine 166 in green.

The mutation studies mentioned above show that multimerization of CIP4 is necessary for tubule formation.

Section 4: Protein-Membrane Interactions In Endocytosis

Learning Objectives:

  1. Identify amino acid residues that are likely to interact with the membrane electrostatically
  2. Understand how the structure of a protein leads to its function
  3. State experimental methods used to determine what amino acids are necessary in cellular processes

In order to begin thinking about how CIP4 functions in endocytosis, recall that membranes are made of phospholipid bilayers. Phospholipids consist of hydrophobic tails that aggregate in the middle of the bilayer and negatively charged hydrophilic phosphate head groups that make up the membrane surfaces. CIP4 has many positively charged amino acid residues on the surface of its F-BAR dimer. In endocytosis, the F-BAR dimer of CIP4 interacts electrostatically with the invaginating membrane because of the attraction of these opposite charges6.

Click Here When clicked, this will show all of the positive residues on the F-BAR dimer.

However, not all of the positive residues located in the F-BAR domain play a role in membrane interactions. Because CIP4 creates tubular vesicles, it is reasonable to predict that the residues important for endocytosis would likely be on the concave surface of F-BAR, allowing it to form the tubular vesicle by constraining the membrane to match the curvature of the dimer. To test this hypothesis, point mutations of these positive residues were performed to see which ones play a role in endocytosis and which ones do not2.

Click Here When clicked, this will show the positive residues that have a role in endocytosis.

Notice that these residues are in four distinct clusters. Researchers found that these residues play a role in endocytosis because upon their mutation, membrane binding of CIP4 and tubule formation was compromised2. Another study found that these clusters of positively charged amino acids could be accurately superimposed on a 3D reconstruction of a cryo-electron microscopic map of the interaction of CIP4 with curved membranes6 (Figure 6).

Figure 6. Top image shows a reconstruction made from a cryo-electron microscopic (cryo-EM) map of the points of binding between the F-BAR dimer and the membrane. F-BAR dimer is in blue-grey and the underlying membrane is in green. The hydrophobic core of the bilayer is 26 angstroms thick. Four clearly resolved points of membrane binding are shown between the F-BAR dimer and the cytoplasmic side of the membrane. The bottom image superimposes the crystal structure of the F-BAR dimer on the cryo-EM image from the top, highlighting the amino acids located at these points in blue6.

References

  1. Strausberg,R.L., Feingold,E.A., Grouse,L.H., Derge,J.G., Klausner,R.D., Collins,F.S., Wagner,L., Shenmen,C.M., Schuler,G.D., Altschul,S.F., Zeeberg,B., Buetow,K.H., Schaefer,C.F., Bhat,N.K., Hopkins,R.F., Jordan,H., Moore,T., Max,S.I., Wang,J., Hsieh,F., Diatchenko,L., Marusina,K., Farmer,A.A., Rubin,G.M., Hong,L., Stapleton,M., Soares,M.B., Bonaldo,M.F., Casavant,T.L., Scheetz,T.E., Brownstein,M.J., Usdin,T.B., Toshiyuki,S., Carninci,P., Prange,C., Raha,S.S., Loquellano,N.A., Peters,G.J., Abramson,R.D., Mullahy,S.J., Bosak,S.A., McEwan,P.J., McKernan,K.J., Malek,J.A., Gunaratne,P.H., Richards,S., Worley,K.C., Hale,S., Garcia,A.M., Gay,L.J., Hulyk,S.W., Villalon,D.K., Muzny,D.M., Sodergren,E.J., Lu,X., Gibbs,R.A., Fahey,J., Helton,E., Ketteman,M., Madan,A., Rodrigues,S., Sanchez,A., Whiting,M., Madan,A., Young,A.C., Shevchenko,Y., Bouffard,G.G., Blakesley,R.W., Touchman,J.W., Green,E.D., Dickson,M.C., Rodriguez,A.C., Grimwood,J., Schmutz,J., Myers,R.M., Butterfield,Y.S., Krzywinski,M.I., Skalska,U., Smailus,D.E., Schnerch,A., Schein,J.E., Jones,S.J. and Marra,M.A. 2002. Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proceedings of the National Academy of Sciences 99(26): 16899-16903
  2. Shimada A, Niwa H, Tsujita K, Suetsugu S, Nitta K, Hanawa-Suetsugu K, Akasaka R, Nishino Y, Toyama M, Chen L, Liu ZJ, Wang BC, Yamamoto M, Terada T, Miyazawa A, Tanaka A, Sugano S, Shirouzu M, Nagayama K, Takenawa T, Yokoyama S. 2007. Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis. Cell 129: 761-772
  3. Miyamoto, K., Tomizawa, T., Koshiba, S., Inoue, M., Kigawa, T., Yokoyama, S. To be Published. Solution structure of the SH3 domain of the Cdc42-interacting protein 4.
  4. Kobashigawa, Y., Kumeta, H., Kanoh, D., Inagaki, F. 2009. The NMR structure of the TC10- and Cdc42-interacting domain of CIP4. Journal of Biomolecular NMR 44: 113-118
  5. Masuda, M. and Mochizuki, N. 2010. Structural characteristics of BAR domain superfamily to sculpt the membrane. Seminars in Cell and Developmental Biology 21: 391-398
  6. Frost A, Perera R, Roux A, Spasov K, Destaing O, Egelman EH, De Camilli P, Unger VM. 2008. Structural basis of membrane invagination by F-BAR domains. Cell 132: 807-817