Research

Chris’ work

Exploration of SUMO-SENP2 interactions

using a high-throughput computational protocol.

Chris A. Kieslich, Jiayu Liao, Dimitrios Morikis

Department of Bioengineering

University of California, Riverside

Sumoylation of cellular proteins by the ubiquitin-like protein, SUMO, has been found to be one of the essential regulation mechanisms in signal transduction and genome integrity. Conjugation by SUMO first requires the maturation of the precursor through the cleaving of a C-terminal peptide. Following SUMO conjugation the SUMO peptide can also be removed from its substrate in vivo. SENP2, an endopeptidase, is responsible for both maturation of SUMO-1 into its conjugatable form, and the deconjugation of SUMO-1 containing species, and therefore plays significant role in the regulation of SUMO-1 sumoylation.

In this study, a high-throughput computational protocol¹ was used to elucidate possible interactions of interest in the SUMO-1:SENP2 complex. The protocol involves: (i) performing a computational Alanine scan, including only ionizable amino acids, (ii) calculation of electrostatic potentials according to the Poisson-Boltzmann equation, (iii) generation of images of spatial distributions of electrostatic potentials, (iv) calculation of electrostatic similarity indices (ESI), using the calculated spatial distributions of electrostatic potentials, and (v) clustering of ESIs. When calculating the free energies of association the contributions of solvation, nonpolar, and Coulombic affects were separated. Finally, the generated clusters were ranked according to calculated free energies of association. A total of 94 Alanine mutants were created and evaluated based on an X-ray structure of the complex formed between the catalytic domain of SENP2 and SUMO-1.²

The data depict important interactions for both SUMO-1:SENP2 binding and the stability of the individual components of the complex. Among the interactions of interest are three intra-molecular and two inter-molecular salt bridges which may be vital to SUMO-1:SENP2 binding and stability. The produced predictions provide physicochemical insight into the mechanism of binding and will be used to guide mutagenesis experiments to gain understanding for binding specificity between SUMO-1 and SENP2.

¹Yang J, Gunopulos D, Morikis D (2007) In Preparation.

²Reverter D, Lima C (2004) Structure

Aliana’s work

Interactions of the V3-loop of HIV-1 gp120 and the N-terminal Domain of CCR5

Aliana López De Victoria, Chris A. Kieslich, Dimitrios Morikis

Department of Bioengineering

University of California, Riverside

HIV-1 involves binding of its envelope glycoprotein gp120 with the CD4 receptor and coreceptors CCR5 or CXCR4 in the host cell. The mechanism of cell infection by HIV is not well understood. The third variable region of gp120 forms a loop, called the V3-loop, which is composed of 31-39 residues. The V3-loop is responsible for determining HIV tropism and plays an important role in viral entry by selecting the appropriate coreceptor. Previous studies have demonstrated that the V3-loop interacts with the N-terminal extra-cellular domain of CCR5 (CCR5-Nt) and that electrostatics play the dominant role in this interaction.^1,2 The electrostatic attraction involves a highly positive V3-loop and a highly negative CCR5-Nt. The overall charge of CCR5-Nt is enhanced by the presence of a minimum of two sulfated tyrosines.

The V3-loop is excessively positively charged and is closed by a disulfide bridge formed by two cysteines. The V3-loop consists of three distinct regions: the base (closer to the core of the protein), the tip at the opposite end, and the stem between the base and the tip. Currently, there are two X-ray structures of gp120 complexes with the V3-loop intact, in which the V3-loop shows structural variability.^3,4 We have applied our high-throughput computational methodology⁵ to delineate the contributions of each ionizable amino acid in the overall electrostatic potential generated by the V3-loop charges, using models from the available X-ray structures. We will discuss the calculated clusters with respect to visualized spatial distributions of electrostatic potentials and their importance for CCR5-Nt binding. We will also present docking models for the V3-loop:CCR5-Nt complex and calculated electrostatic and nonpolar free energies of association. These data will form the basis for future design of agonists and antagonists for the V3-loop:CCR5-Nt interaction.

¹Morikis D, Rizos AK, Spandidos DA, Krambovitis E (2007) International Journal of Molecular Medicine 19:343-5.

²Rizos AK, Tsikalas I, Morikis D, Galanakis P, SpyrouliasGA, Krambovitis E (2006) Journal of Non-Crystalline Solids 352:4451-58.

³Huang C, Lam SN, Acharya P, Tang M, Xiang SH, Hussan SS, Stanfield RL, Robinson J, Sodroski J, Wilson IA, Wyatt R, Bewly CA, Kwong PD (2007) Science 317:1930-34.

⁴Huang CC, Tang M, Zhang MY, Majeed S, Montabana E, Stanfielg Rl, Dimitrov DS, Korber B, Sodroski J, Wilson IA, Wyatt R, Kwong PD (2005) Science 310:1025-28.

⁵Yang J, Gunopulos D, Morikis D (2008) In Preparation.

Alex’s work

Computational Prediction of Association Free Energies for the C3d:CR2 Complex and Comparison to Experimental Data

Alexander S. Cheung,¹ Jianfeng Yang,² Chris A. Kieslich,¹ Dimitrios Morikis¹

Department of ¹Bioengineering and ²Computer Science and Engineering

University of California, Riverside

The complement system is part of the innate immune system and functions to clear pathogenic threats. The association between complement protein C3d and B or T cell-receptor CR2 (complement receptor 2) represents a crucial link between innate and adaptive immunities. The goal of this study is to computationally predict association abilities of C3d and CR2 mutants by theoretically calculating electrostatic free energies of association with and without solvation effects. We demonstrate that incorporation of solvation effects is necessary to accurately predict previously published experimental data for the association abilities (relative to the parent proteins) of specific C3d and CR2 mutants. We show that a proportional relationship exists between the predicted solvation free energy differences and the experimental data. Additionally, an inversely proportional relationship is demonstrated between the calculated solvation free energy differences and previously calculated ionization free energy differences. Changes in the nonpolar free energies upon association were also calculated, but for the mutants considered in this study, are negligible. Our results yield new insights into the physicochemical properties underlying C3d:CR2 association. Our results can also be extended to any complex with excessively charged components. This basic study contributes toward developing a theoretical understanding of immune system regulation at the molecular level, and can be the groundwork for the design of regulators with tailored properties, vaccines, and biotechnology products in general.

Soon to come research updates from Homero and Gabrielle.

HOME