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The solution structure of the complex of enzyme IIA of the N,N'-diacetylchitobiose (Chb) transporter with the histidine phosphocarrier protein HPr has been solved by NMR. The IIAChb-HPr complex completes the structure elucidation of representative cytoplasmic complexes for all four sugar branches of the bacterial phosphoryl transfer system (PTS). The active site His-89 of IIAChb was mutated to Glu to mimic the phosphorylated state. IIAChb(H89E) and HPr form a weak complex with a KD of ∼0.7 mM. The interacting binding surfaces, concave for IIAChb and convex for HPr, complement each other in terms of shape, residue type, and charge distribution, with predominantly hydrophobic residues, interspersed by some uncharged polar residues, located centrally, and polar and charged residues at the periphery. The active site histidine of HPr, His-15, is buried within the active site cleft of IIAChb formed at the interface of two adjacent subunits of the IIAChb trimer, thereby coming into close proximity with the active site residue, H89E, of IIAChb. A His89-P-His-15 pentacoordinate phosphoryl transition state can readily be modeled without necessitating any significant conformational changes, thereby facilitating rapid phosphoryl transfer. Comparison of the IIAChb-HPr complex with the IIAChb-IIBChb complex, as well as with other cytoplasmic complexes of the PTS, highlights a unifying mechanism for recognition of structurally diverse partners. This involves generating similar binding surfaces from entirely different underlying structural elements, large interaction surfaces coupled with extensive redundancy, and side chain conformational plasticity to optimize diverse sets of intermolecular interactions. [ABSTRACT FROM AUTHOR]

The solution structure of the IIA-IIB complex of the N,N'- diacetylchitobiose (Chb) transporter of the Escherichia coli phosphotransferase system has been solved by NMR. The active site His-89 of IIAChb was mutated to Glu to mimic the phosphorylated state and the active site Cys-10 of IIBChb was substituted by serine to prevent intermolecular disulfide bond formation. Binding is weak with a KD of ∼1.3 mM. The two complementary interaction surfaces are largely hydrophobic, with the protruding active site loop (residues 9-16) of IIBChb buried deep within the active site cleft formed at the interface of two adjacent sub-units of the IIAChb trimer. The central hydrophobic portion of the interface is surrounded by a ring of polar and charged residues that provide a relatively small number of electrostatic intermolecular interactions that serve to correctly align the two proteins. The conformation of the active site loop in unphosphorylated IIBChb is inconsistent with the formation of a phosphoryl transition state intermediate because of steric hindrance, especially from the methyl group of Ala-12 of IIBChb. Phosphorylation of IIBChb is accompanied by a conformational change within the active site loop such that its path from residues 11-13 follows a mirror-like image relative to that in the unphosphorylated state. This involves a transition of the ϕ/ψ angles of Gly-13 from the right to left α-helical region, as well as smaller changes in the backbone torsion angles of Ala-12 and Met-14. The resulting active site conformation is fully compatible with the formation of the His-89-P-Cys-10 phosphoryl transition state without necessitating any change in relative translation or orientation of the two proteins within the complex. [ABSTRACT FROM AUTHOR]

In this paper, we present a series of heteronuclear NMR experiments for the direct observation and characterization of lysine NH3 groups in proteins. In the context of the HoxD9 homeodomain bound specifically to DNA we were able to directly observe three cross-peaks, arising from lysine NH3 groups, with 15N chemical shifts around ~33 ppm at pH 5.8 and 35 °C. Measurement of water-exchange rates and various types of 15N transverse relaxation rates for these NH3 groups, reveals that rapid water exchange dominates the 15N relaxation for antiphase coherence with respect to ¹H through scalar relaxation of the second kind. As a consequence of this phenomenon, 15N line shapes of NH3 signals in a conventional ¹H15N heteronuclear single quantum coherence (HSQC) correlation experiment are much broader than those of backbone amide groups. A 2D ¹H-15N correlation experiment that exclusively observes in-phase 15N transverse coherence (termed HISOC for heteronuclear in-phase single quantum coherence spectroscopy) is independent of scalar relaxation in the t1 (15N) time domain and as a result exhibits strikingly sharper 15N line shapes and higher intensities for NH3 cross-peaks than either HSQC or heteronuclear multiple quantum coherence (HMQC) correlation experiments. Coherence transfer through the relatively small J-coupling between 15Nζ and 13Cϵ (4.7-5.0 Hz) can be achieved with high efficiency by maintaining in-phase 15N coherence owing to its slow relaxation. With the use of a suite of triple resonance experiments based on the same design principles as the HISQC, all the NH3 cross-peaks observed in the HISQC spectrum could be assigned to lysines that directly interact with DNA phosphate groups. Selective observation of functional NH3 groups is feasible because of hydrogen bonding or salt bridges that protect them from rapid water exchange. Finally, we consider the potential use of lysine NH3 groups as an alternative probe for larger systems as illustrated by data obtained on the 128-kDa enzyme I dimer. [ABSTRACT FROM AUTHOR]

In idiopathic Parkinson's disease, intracytoplasmic neuronal inclusions (Lewy bodies) containing aggregates of the protein α-synuclein (αS) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded, αS to neurotoxic forms are largely unknown. Using paramagnetic relaxation enhancement and NMR dipolar couplings, we show that monomeric αS assumes conformations that are stabilized by. long-range interactions and act to inhibit oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to microseconds corresponding to the time scale of secondary structure formation during folding. Polyamine binding and/or temperature increase, conditions that induce aggregation in vitro, release this inherent tertiary structure, leading to a completely unfolded conformation that associates readily. Stabilization of the native, autoinhibitory structure of αS constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease. [ABSTRACT FROM AUTHOR]