Nmr Studies Of The Regulation Of Cell Signaling

National Heart, Lung, And Blood Institute

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Abstract

As a progression in developing new technology to characterize dynamic molecular events which regulate important biological function, we chose to look at retroviral capsid assembly. This protein is a part of the Gag-poly protein which is processed as part of the maturation of the virus. The assembly and disassembly of the capsid particle is crucial for viral budding from and entry into the host cell, respectively. We showed that capsid assembly occurs due to two types of distinct molecular interactions. The N-terminal beta hairpin promotes the elongation of helix 1 which forms the oligomerization interface of the capsid particle. This event occurs at a slower timescale than the dimerization that involves the C-terminal domain of the capsid. We could only established the above observations by using a barrage of NMR experiments. This is largely due to the dynamic nature of the molecular interactions. We also synthesized a compound (methylated-DOTA) that can coordinate lanthanide ligand with reduced flexibility. This was done in collaborating with the Imaging Probe Development Group. The goal was to achieve a substantial increase in observable Pseudo Contact Shift (PCS) and use the information for structure determination. In addition we also showed that the methylated-DOTA-lanthanide adopts two isomers. The populations of these isomers depend on the size of the lanthanide metal being coordinated. The population ratios that we measured by observing PCS on a protein matched those obtained from HPLC on the methylated-DOTA-lanthanide. We carried out temperature dependence study on the DOTA lanthanide to show that the size of the susceptibility tensor depends highly on temperature and this is due to bound water exchange rate. The slower the rate the larger the tensor. We showed that the methylated-DOTA-lanthanide is also very practical in studying intrinsically disordered proteins by introducing PCS which results in better dispersion of the typically overlapping NMR resonances. Moreover, in the case of dynamic protein-proton complexes, such as those that exhibit encounter complexes, using spin label nitroxide to get structural information can be complicated by the encounter complexes. On the other hand, we showed that by using the PCS we can determine the major form of the complex between Enzyme I and NPr of the nitrogen transfer system. Using the above unique approach we have been able to establish that encounter complexes between two paralogous systems can compete against each other. We monitored changes in Enzyme I and NPr specific and encounter complexes in the presence of HPr. We previously established that HPr doesn't interact specifically with Enzyme I. With increasing HPr concentration we showed that NPr encounter can be modulated such that the specific Enzyme I and NPr complex population is increased, effectively increasing the affinity of the complex. This is a surprising finding, therefore we decided to follow up this study with a modeling study which we could recapitulate the encounter profile between NPr and enzyme I in the presence of HPr. This study reveals the lack of understanding beyond competition of specific substrate to regulate biological function. We looked at another weak protein-protein interaction that has biological relevance. In the case of Tsg101, its interaction with ubiquitin (Ub) is rather weak. We were able to detect this interaction with the new paramagnetic technology that we developed above. We established an inhibitor to this Ub-Tsg101 interaction that has allowed us to decipher the Ub signaling in viral trafficking in the host sell. In addition, using NMR we identified another Ub binding site on Tsg101. We confirmed that Tsg101 recognized Di-Ubiquitin (Di-Ub), specifically linked at K63. We also have been able to determine using NMR that Di-Ub binds Tsg101 in two distinct sites. These two sites have different physiological consequences. One site, the so called vestigial Ub binding site controls recruitment of Tsg101 by Hiv-1 Gag to the plasma membrane, while the N-termninal Ub site seems to be correlated to the nuclear capsid determinant of trafficking Hiv-1 Gag. Interestingly, tri-Ub doesn't bind as well as Di-Ub to Tsg101. This finding is novel and allows us to distinguish multiple facets to Ub signaling in the Tsg101 (ESCRTI) pathway. We have continue to use our finding of Tsg101 and Ub interaction to develop small molecules that can inhibit their interaction. We found a family of prazoles, which have been clinically used as proton pump inhibitors, can inhibit Tsg101 and Ub interaction. We showed that this inhibition can interfere with HIV-1 virus particle release from host cells. Moreover, this inhibition also seems to reduce viral protein production, such as Gag, in the host cells. We showed that the same effect could be observed for other viruses, therefore signaling the potential for the prazole family of compounds as broad antiviral agents. Since we know exactly the molecular mechanism for Tsg101-Ub inhibition, we can use this knowledge to push for new generation of compounds that can provide more specificity and potency. This effort has expanded to test our compounds against other family of viruses, including Coronavirus. This was done through the NIAID cores for anti-viral testing. A couple of our compounds showed efficacy to block these viruses, including SARS-CoV2. We are generating new derivatives of our compounds to try to improve their selectivity. In parallel we are also investigating the reason why cells would create a pseudo-E2-ubiquitin Ligase, which is what Tsg101 is. It has the same structure as Ub E2 ligase with the catalytic cysteine replaced by a tyrosine, thus Tsg101 has no enzymatic activity. Furthermore, we also recently showed that this protein can recognize cellular RNA. This ability somehow is linked to Ub recognition in the cell. Th next phase of our research is directed towards combining all of our findings to draw a general scheme of how all of these processes are tied together to benefit cell trafficking and how viruses can modify them for their replication. We expanded our study on Tsg101 by looking at its potential interaction with Nedd4, and E3 ubiquitin ligase shown to be important for HIV-1 replication. We showed that Tsg101 UEV domain does interact with the HECT domain of the Nedd4. This interaction is quite weak. We used NMR PRE experiment to identify regions in HECT that contact Tsg101. Those residues involved in this interaction belong to the hinge region between the N- and C-lobe of HECT. We validated this finding using a rescue assay in the HIV-1 virus-like-particle release and Nedd4 mutations in the sites identified by our NMR experiment. In one of our assays to validate that the a1-helix of Nedd4 was important for its enzyme activation, we noticed that the western band in the pull down assay to identify Tsg101 and Nedd4 complex was significantly higher than what we usually observed. After further testing we showed that the level of Nedd4 is higher when more Tsg101 is present. We were able to also showed that Nedd4 gets poly-ubiquinated and degraded when Tsg101 is not present. Thus Tsg101 serves as a chaperon for Nedd4 in the cell. More over the reverse is also true, when Nedd4 is present, Tsg101 half life is also longer. There is definitely a direct interplay between these two proteins that involves ubiquitin signaling that at the end regulates their level. In addition to the above studies, we also tested the unique signaling peptide for Tsg101 interaction PTAP (Pro-The-Ala-Pro) which is all found in the protein Hrs, part of ESCRT-0, as well as in CoV2 genome (PTEP, PTQP, PNQP). We could identify a second recognition site which is specific to PTEP in Tsg101. We are carrying out hybrid (CoV2-HIV-1) cell assay to validate our findings.