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Human adenylate kinase 1 (<i>h</i>AK1) plays a vital role in the energetic and metabolic regulation of cell life, and impaired functions of <i>h</i>AK1 are closely associated with many diseases. In the presence of Mg²⁺ ions, <i>h</i>AK1 in vivo can catalyze two ADP molecules into one ATP and one AMP molecule, activating the downstream AMP signaling. The ADP-binding also initiates AK1 transition from an open conformation to a closed conformation. However, how substrate binding triggers the conformational transition of <i>h</i>AK1 is still unclear, and the underlying molecular mechanisms remain elusive. Herein, we determined the solution structure of apo-<i>h</i>AK1 and its key residues for catalyzing ADP, and characterized backbone dynamics characteristics of apo-<i>h</i>AK1 and <i>h</i>AK1-Mg²⁺-ADP complex (holo-<i>h</i>AK1) using NMR relaxation experiments. We found that ADP was primarily bound to a cavity surrounded by the LID, NMP, and CORE domains of <i>h</i>AK1, and identified several critical residues for <i>h</i>AK1 catalyzing ADP including G16, G18, G20, G22, T39, G40, R44, V67, D93, G94, D140, and D141. Furthermore, we found that apo-<i>h</i>AK1 adopts an open conformation with significant ps-ns internal mobility, and Mg²⁺-ADP binding triggered conformational transition of <i>h</i>AK1 by suppressing the ps-ns internal motions of α₃α₄ in the NMP domain and α₇α₈ in the LID domain. Both α₃α₄ and α₇α₈ fragments became more rigid so as to fix the substrate, while the catalyzing center of <i>h</i>AK1 experiences promoted µs-ms conformational exchange, potentially facilitating catalysis reaction and conformational transition. Our results provide the structural basis of <i>h</i>AK1 catalyzing ADP into ATP and AMP, and disclose the driving force that triggers the conformational transition of <i>h</i>AK1, which will deepen understanding of the molecular mechanisms of <i>h</i>AK1 functions.