The effect of cellular crowding environments on protein structure and stability is a key issue in molecular and cellular biology. The classical view of crowding emphasizes the volume exclusion effect that generally favors compact, native states. Here, results from molecular dynamics simulations and NMR experiments show that protein crowders may destabilize native states via protein-protein interactions. In the model system considered here, mixtures of villin head piece and protein G at high concentrations, villin structures become increasingly destabilized upon increasing crowder concentrations. The denatured states observed in the simulation involve partial unfolding as well as more subtle conformational shifts. The unfolded states remain overall compact and only partially overlap with unfolded ensembles at high temperature and in the presence of urea. NMR measurements on the same systems confirm structural changes upon crowding based on changes of chemical shifts relative to dilute conditions. An analysis of protein-protein interactions and energetic aspects suggests the importance of enthalpic and solvation contributions to the crowding free energies that challenge an entropic-centered view of crowding effects.

The recent expansion of structural genomics has increased the demands for quick and accurate protein structure determination by NMR spectroscopy. The conventional strategy without an automated protocol can no longer satisfy the needs of high-throughput application to a large number of proteins, with each data set including many NMR spectra, chemical shifts, NOE assignments, and calculated structures. We have developed the new software KUJIRA, a package of integrated modules for the systematic and interactive analysis of NMR data, which is designed to reduce the tediousness of organizing and manipulating a large number of NMR data sets. In combination with CYANA, the program for automated NOE assignment and structure determination, we have established a robust and highly optimized strategy for comprehensive protein structure analysis. An application of KUJIRA in accordance with our new strategy was carried out by a non-expert in NMR structure analysis, demonstrating that the accurate assignment of the chemical shifts and a high-quality structure of a small protein can be completed in a few weeks. The high completeness of the chemical shift assignment and the NOE assignment achieved by the systematic analysis using KUJIRA and CYANA led, in practice, to increased reliability of the determined structure.

Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes specifically the pSer/pThr-Pro motif. The cis-trans isomerization mechanism has been studied by various approaches, including X-ray crystallography, site-directed mutagenesis, and the kinetic isotope effect on isomerization. However, a complete picture of the reaction mechanism remains elusive. On the basis of the X-ray structure of Pin1, residue C113 was proposed to play a nucleophile attacker to catalyze the isomerization. The controversial result that the C113D Pin1 mutant retains the activity, albeit at a reduced level, challenges the importance of C113 as a catalyst. To facilitate our understanding of the Pin1 isomerization process, we compared the structures and dynamics of the wild type with those of the C113D mutant Pin1 PPIase domains (residues 51-163). We found the C113D mutation disturbed the hydrogen bonds between the conserved histidine residues, H59 and H157 ("dual-histidine motif"); H59 imidazole forms a stable hydrogen bond to H157 in the wild type, whereas it has a strong hydrogen bond to D113 with weakened bonding to H157 in the C113D mutant. The C113D mutation unbalanced the hydrogen bonding tug of war for H59 between C113/D113 and H157 and destabilized the catalytic site structure, which eventually resulted in an altered conformation of the basic triad (K63, R68, and R69) that binds to the phosphate group in a substrate. The change in the basic triad structure could explain the severely weakened substrate binding ability of the C113D mutant. Overall, this work demonstrated that C113 plays a role in keeping the catalytic site in an active fold, which has never before been described.

TRIM5␣ rh is a cytosolic protein that potently restricts HIV-1 before reverse transcription. TRIM5␣ rh is composed of four different domains: RING, B-box 2, coiled coil, and B30.2(SPRY). The contribution of each of these domains to restriction has been extensively studied, with the exception of the RING domain. The RING domain of TRIM5␣ exhibits E3-ubiquitin ligase activity, but the contribution of this activity to the restriction of HIV-1 is not known. To test the hypothesis that the E3-ubiquitin ligase activity of the RING domain modulates TRIM5␣ rh restriction of HIV-1, we correlated the E3-ubiquitin ligase activity of a panel of TRIM5␣ rh RING domain variants with the ability of these mutant proteins to restrict HIV-1. For this purpose, we first solved the nuclear magnetic resonance structure of the RING domain of TRIM5␣ and defined potential functional regions of the RING domain by homology to other RING domains. With this structural information, we performed a systematic mutagenesis of the RING domain regions and tested the TRIM5␣ RING domain variants for the ability to undergo self-ubiquitylation. Several residues, particularly the ones on the E2-binding region of the RING domain, were defective in their self-ubiquitylation ability. To correlate HIV-1 restriction to self-ubiquitylation, we used RING domain mutant proteins that were defective in self-ubiquitylation but preserve important properties required for potent restriction by TRIM5␣ rh , such as capsid binding and higher-order self-association. From these investigations, we found a set of residues that when mutated results in TRIM5␣ molecules that lost both the ability to potently restrict HIV-1 and their self-ubiquitylation activity. Remarkably, all of these changes were in residues located in the E2-binding region of the RING domain. Overall, these results demonstrate a role for TRIM5␣ self-ubiquitylation in the ability of TRIM5␣ to restrict HIV-1.Several newly discovered proteins that are endogenously expressed in primates show the ability to dominantly block retroviral infection and cross-species transmission by interfering with the early phase of viral replication (3,33,57,63). Of particular interest are members of the tripartite motif (TRIM) family of proteins. Splicing variant alpha of TRIM5 from rhesus macaques (TRIM5␣ rh ) is an ϳ53-kDa cytosolic protein that potently restricts HIV-1 (28, 61). TRIM5␣ rh blocks HIV-1 and certain other retroviruses soon after viral entry but prior to reverse transcription (28, 63). The retroviral capsid protein is the viral determinant for susceptibility to restriction by TRIM5␣ (48). Studies on the fate of the HIV-1 capsid in the cytosol of infected cells have correlated restriction with a decrease amount of cytosolic particulate capsid (11,51,64).TRIM5␣ rh is composed of four different domains: RING, B-box 2, coiled coil, and B30.2(SPRY) (53). The RING domain of TRIM5␣ rh is an E3-ubiquitin ligase (13,27,37,43,68); however, a role for the really interesting new gene (RING) domain's E3-ubiquitin ligase activ...

Vaccinia-related kinase 1 (VRK1) is one of the mitotic kinases that play important roles in cell cycle, nuclear condensation, and transcription regulation. Kinase domain structures of two other VRK family members (VRK2 and VRK3) have been determined previously. However, the structure of VRK1, the most extensively studied and constitutively active VRK member, is yet to be characterized. Here, we present the nuclear magnetic resonance (NMR) solution structure of a catalytically active form of human VRK1 with its extended C-terminal tail (residues 1-361). The NMR structure of human VRK1 reveals that the C-terminal tail orients toward the catalytic site and forms a number of interactions that are critical for structural stability and catalysis. The role of this unique C-terminal tail was further investigated by deletion mutant studies where deletion of the terminal tail resulted in a dramatic reduction in the autocatalytic activity of VRK1. NMR titration studies carried out with ATP or an ATP analog confirm that ATP/ATP analogs interact with all of the crucial residues present in important motifs of the protein kinase such as the hinge region, catalytic loop, DYG motif, and thereby suggest that the catalytic domain of VRK1 is not atypical. In addition to the conventional interactions, some of the residues present on the extended C-terminal tail also interact with the ligands. These observations also substantiate the role of the extended C-terminal tail in the biological activity of VRK1.The coordinated action of protein kinases and phosphatases plays a major role in regulating signal transduction events in a multicellular organism (1-3). Kinases comprise one of the major members of the human genome (4), characterized by the presence of a conserved catalytic domain of approximately 300 amino acids which is involved in the phosphotransfer reactions (5-7). Vaccinia-related kinase 1 (VRK1) 4 belongs to a novel group of serine/threonine kinases that bear a high degree of sequence similarity with vaccinia virus B1 R kinase (8, 9). Three members of VRK family are known in the human genome and show a significant sequence similarity among themselves with respect to their catalytic domains, but differ in their regulatory domains (8, 10). VRK1 is the most well studied member of the family, known to participate in a number of biological activities especially in cellular proliferations as well as in management of cellular stress situations (11). A number of studies demonstrated that VRK1 phosphorylates several stress-related transcription factors e.g. p53, c-Jun, ATF2, which in turn play a major role in regulating cellular fate (10 -12).Several studies have also reported the role of VRK1 in cellular proliferation both in normal cells as well as in cells with uncontrolled proliferation (13). Elevated levels of VRK1 protein have been observed in highly proliferative cell lines, indicating its role in cell division (14). Furthermore, VRK1 is a nuclear protein, known to play an important role in cell cycle progression through phosph...